Covalent diabodies and uses thereof

ABSTRACT

The present invention is directed to diabody molecules and uses thereof in the treatment of a variety of diseases and disorders, including immunological disorders, infectious disease, intoxication and cancers. The diabody molecules of the invention comprise two polypeptide chains that associate to form at least two epitope binding sites, which may recognize the same or different epitopes on the same or differing antigens. Additionally, the antigens may be from the same or different molecules. The individual polypeptide chains of the diabody molecule may be covalently bound through non-peptide bond covalent bonds, such as, but not limited to, disulfide bonding of cysteine residues located within each polypeptide chain. In particular embodiments, the diabody molecules of the present invention further comprise an Fc region, which allows antibody-like functionality to engineered into the molecule.

This application claims the benefit of U.S. Patent Application Ser. No.60/671,657 (filed Apr. 15, 2005; expired); Ser. No. 11/409,339 (filedApr. 17, 2006; pending); PCT/US06/014481 (filed Apr. 17, 2006; expired);60/945,523 (filed Jun. 21, 2007; expired), 61/019,051 (filed Jan. 4,2008; expired); Ser. No. 12/138,867 (filed Jun. 13, 2008, pending),PCT/US08/066,957 (filed Jun. 13, 2008, pending), 61/139,352 (filed Dec.19, 2008, pending), 61/156,035 (filed Feb. 27, 2009, pending) and61/256,779 (filed Oct. 30, 2009; pending) all of which applications areherein incorporated by reference in their entireties.

1. FIELD OF THE INVENTION

The present invention is directed to diabody molecules, otherwisereferred to as “dual affinity retargeting reagents” (“DARTS”), and usesthereof in the treatment of a variety of diseases and disorders,including immunological disorders and cancers. The diabody molecules ofthe invention comprise at least two polypeptide chains that associate toform at least two epitope binding sites, which may recognize the same ordifferent epitopes. Additionally, the epitopes may be from the same ordifferent molecules or located on the same or different cells. Theindividual polypeptide chains of the diabody molecule may be covalentlybound through non-peptide bond covalent bonds, such as, but not limitedto, disulfide bonding of cysteine residues located within eachpolypeptide chain. In particular embodiments, the diabody molecules ofthe present invention further comprise an Fc region, which allowsantibody-like functionality to be engineered into the molecule.

2. BACKGROUND OF THE INVENTION

The design of covalent diabodies is based on the single chain Fvconstruct (scFv) (Holliger et al. (1993) “‘Diabodies’: Small BivalentAnd Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. USA90:6444-6448; herein incorporated by reference in its entirety). In anintact, unmodified IgG, the VL and VH domains are located on separatepolypeptide chains, i.e., the light chain and the heavy chain,respectively. Interaction of an antibody light chain and an antibodyheavy chain and, in particular, interaction of VL and VH domains formsone of the epitope binding sites of the antibody. In contrast, the scFvconstruct comprises a VL and VH domain of an antibody contained in asingle polypeptide chain wherein the domains are separated by a flexiblelinker of sufficient length to allow self-assembly of the two domainsinto a functional epitope binding site. Where self assembly of the isimpossible due to a linker of insufficient length (less than about 12amino acid residues), two of the scFv constructs interact with eachother to form a bivalent molecule, the VL of one chain associating withthe VH of the other (reviewed in Marvin et al. (2005) “RecombinantApproaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. Sin.26:649-658). Moreover, addition of a cysteine residue to the c-terminusof the construct has been show to allow disulfide bonding of thepolypeptide chains, stabilizing the resulting dimer without interferingwith the binding characteristics of the bivalent molecule (see e.g.,Olafsen et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody AllowsSite-Specific Conjugation And Radiolabeling For Tumor TargetingApplications,” Prot. Engr. Des. Sel. 17:21-27). Further, where VL and VHdomains of differing specificity are selected, not only a bivalent, butalso a bispecific molecule may be constructed.

Bivalent diabodies have wide ranging applications including therapy andimmunodiagnosis. Bivalency allows for great flexibility in the designand engineering of diabody in various applications, providing enhancedavidity to multimeric antigens, the cross-linking of differing antigens,and directed targeting to specific cell types relying on the presence ofboth target antigens. Due to their increased valency, low dissociationrates and rapid clearance from the circulation (for diabodies of smallsize, at or below ˜50 kDa), diabody molecules known in the art have alsoshown particular use in the filed of tumor imaging (Fitzgerald et al.(1997) “Improved Tumour Targeting By Disulphide Stabilized DiabodiesExpressed In Pichia pastoris,” Protein Eng. 10:1221). Of particularimportance is the cross linking of differing cells, for example thecross linking of cytotoxic T cells to tumor cells (Staerz et al. (1985)“Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature314:628-631, and Holliger et al. (1996) “Specific Killing Of LymphomaCells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” ProteinEng. 9:299-305). Diabody epitope binding domains may also be directed toa surface determinant of any immune effector cell such as CD3, CD16,CD32, or CD64, which are expressed on T lymphocytes, natural killer (NK)cells or other mononuclear cells. In many studies, diabody binding toeffector cell determinants, e.g., Fcγ receptors (FcγR), was also foundto activate the effector cell (Holliger et al. (1996) “Specific KillingOf Lymphoma Cells By Cytotoxic T-Cells Mediated By A BispecificDiabody,” Protein Eng. 9:299-305; Holliger et al. (1999)“Carcinoembryonic Antigen (CEA)-Specific T-cell Activation In ColonCarcinoma Induced By Anti-CD3 x Anti-CEA Bispecific Diabodies AndB7×Anti-CEA Bispecific Fusion Proteins,” Cancer Res. 59:2909-2916).Normally, effector cell activation is triggered by the binding of anantigen bound antibody to an effector cell via Fc-FcγR interaction;thus, in this regard, diabody molecules of the invention may exhibitIg-like functionality independent of whether they comprise an Fc domain(e.g., as assayed in any effector function assay known in the art orexemplified herein (e.g., ADCC assay)). By cross-linking tumor andeffector cells, the diabody not only brings the effector cell within theproximity of the tumor cells but leads to effective tumor killing (seee.g., Cao et al. (2003) “Bispecific Antibody Conjugates InTherapeutics,” Adv. Drug. Deliv. Rev. 55:171-197, hereby incorporated byreference herein in its entirety).

2.1 Effector Cell Receptors and their Roles in the Immune System

In traditional immune function the interaction of antibody-antigencomplexes with cells of the immune system results in a wide array ofresponses, ranging from effector functions such as antibody-dependentcytotoxicity, mast cell degranulation, and phagocytosis toimmunomodulatory signals such as regulating lymphocyte proliferation andantibody secretion. All these interactions are initiated through thebinding of the Fc domain of antibodies or immune complexes tospecialized cell surface receptors on hematopoietic cells. The diversityof cellular responses triggered by antibodies and immune complexesresults from the structural heterogeneity of Fc receptors. Fc receptorsshare structurally related an antigen binding domains which presumablymediate intracellular signaling.

The Fcγ receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fcγ portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe alpha chain of the Fcγ receptor. Fcγ receptors are defined by theirspecificity for immunoglobulin subtypes. Fcγ receptors for IgG arereferred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Differentaccessory cells bear Fcγ receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. (1991) “FcReceptors,” Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. (2001)“Stimulatory And Inhibitory Signals Originating From The MacrophageFcgamma Receptors,” Microbes and Infection, 3: 131-139; Billadeau D. D.et al. (2002), “ITAMs Versus ITIMs: Striking A Balance During CellRegulation,” The Journal of Clinical Investigation, 2(109): 161-1681;Ravetch J. V. et al. (2000) “Immune Inhibitory Receptors,” Science, 290:84-89; Ravetch J. V. et al., (2001) “IgG Fc Receptors,” Annu. Rev.Immunol. 19:275-90; Ravetch J. V. (1994) “Fc Receptors: Rubor Redux,”Cell, 78(4): 553-60). The different Fcγ receptors, the cells thatexpress them, and their isotype specificity is summarized in Table 1(adapted from Immunobiology: The Immune System in Health and Disease,4^(th) ed. 1999, Elsevier Science Ltd/Garland Publishing, New York).

Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologybetween the three family members suggest they arose from a commonprogenitor perhaps by gene duplication.

FcγRII(CD32)

FcγRII proteins are 40 kDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogenous responses to receptorligation. The fundamental difference is that the A isoform initiatesintracellular signaling leading to cell activation such as phagocytosisand respiratory burst, whereas the B isoform initiates inhibitorysignals, e.g., inhibiting B-cell activation.

FcγRIII (CD16)

Due to heterogeneity within this class, the size of FcγRIII rangesbetween 40 and 80 kDa in mouse and man. Two human genes encode twotranscripts, FcγRIIIA, an integral membrane glycoprotein, and FcγRIIIB,a glycosylphosphatidyl-inositol (GPI)-linked version. One murine geneencodes an FcγRIII homologous to the membrane spanning human FcγRIIIA.The FcγRIII shares structural characteristics with each of the other twoFcγRs. Like FcγRII, FcγRIII binds IgG with low affinity and contains thecorresponding two extracellular Ig-like domains. FcγRIIIA is expressedin macrophages, mast cells and is the lone FcγR in NK cells. TheGPI-linked FcγRIIIB is currently known to be expressed only in humanneutrophils.

Signaling through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When co-ligatedalong with an activating FcγR, the ITIM in FcγRIIB becomesphosphorylated and attracts the SH2 domain of the inosital polyphosphate5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengersreleased as a consequence of ITAM-containing FcγR-mediated tyrosinekinase activation, consequently preventing the influx of intracellularCa⁺⁺. Thus crosslinking of FcγRIIB dampens the activating response toFcγR ligation and inhibits cellular responsiveness. B cell activation, Bcell proliferation and antibody secretion is thus aborted.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotypes ReceptorBinding Cell Type Effect of Ligation FcγRI (CD64) IgG1 MacrophagesUptake Stimulation 10⁸ M⁻¹ Neutrophils Activation of respiratoryEosinophils burst Dendritic cells Induction of killing FcγRII-A IgG1Macrophages Uptake Granule Release (CD32) 2 × 10⁶ M⁻¹ NeutrophilsEosinophils Dendritic cells Platelets Langerhan cells FcγRII-B2 IgG1Macrophages Uptake Inhibition of (CD32) 2 × 10⁶ M⁻¹ NeutrophilsStimulation Eosinophils FcγRII-B1 IgG1 B cells No Uptake (CD32) 2 × 10⁶M⁻¹ Mast cells Inhibition of Stimulation FcγRIII IgG1 NK cells Inductionof Killing (CD16) 5 × 10⁵ M⁻¹ Eosinophil Macrophages Neutrophils MastCells FcεRI IgE Mast cells Secretion of granules 10¹⁰ M⁻¹ EosinophilBasophils FcαRI IgA1, IgA2 Macrophages Uptake Induction of (CD89) 10⁷M⁻¹ Neutrophils Killing Eosinophils

3. SUMMARY OF THE INVENTION

The present invention relates to covalent diabodies and/or covalentdiabody molecules and to their use in the treatment of a variety ofdiseases and disorders including cancer, autoimmune disorders, allergydisorders and infectious diseases caused by bacteria, fungi or viruses.Preferably, the diabody of the present invention can bind to twodifferent epitopes on two different cells wherein the first epitope isexpressed on a different cell type than the second epitope, such thatthe diabody can bring the two cells together.

In one embodiment, the present invention is directed to a covalentbispecific diabody, which diabody comprises a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope, and,optionally, (iii) a third domain comprising at least one cysteineresidue, which first and second domains are covalently linked such thatthe first and second domains do not associate to form an epitope bindingsite; which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and, optionally, (iii) a sixth domain comprising at least onecysteine residue, which fourth and fifth domains are covalently linkedsuch that the fourth and fifth domains do not associate to form anepitope binding site; and wherein the first polypeptide chain and thesecond polypeptide chain are covalently linked, with the proviso thatthe covalent link is not a peptide bond; wherein the first domain andthe fifth domain associate to form a first binding site (VL1)(VH1) thatbinds the first epitope; wherein the second domain and the fourth domainassociate to form a second binding site (VL2)(VH2) that binds the secondepitope.

In another embodiment, the present invention is directed to a covalentbispecific diabody, which diabody comprises a first and a secondpolypeptide chain, which first polypeptide chain comprises (1) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope and (iii) athird domain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; which secondpolypeptide chain comprises (i) a fourth domain comprising a bindingregion of a light chain variable domain of the second immunoglobulin(VL2), (ii) a fifth domain comprising a binding region of a heavy chainvariable domain of the first immunoglobulin (VH1), which fourth andfifth domains are covalently linked such that the third and fourthdomains do not associate to form an epitope binding site; and whereinthe first polypeptide chain and the second polypeptide chain arecovalently linked, with the proviso that the covalent link is not apeptide bond; wherein the first domain and the fifth domain associate toform a first binding site (VL1)(VH1) that binds the first epitope;wherein the second domain and the fourth domain associate to form asecond binding site (VL2)(VH2) that binds the second epitope.

In certain aspects, the present invention is directed to diabodymolecule, which molecule comprises a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; which secondpolypeptide chain comprises (i) a fourth domain comprising a bindingregion of a light chain variable domain of the second immunoglobulin(VL2), (ii) a fifth domain comprising a binding region of a heavy chainvariable domain of the first immunoglobulin (VH1), and (iii) a sixthdomain comprising at least one cysteine residue, which fourth and fifthdomains are covalently linked such that the fourth and fifth domains donot associate to form an epitope binding site; and wherein the firstpolypeptide chain and the second polypeptide chain are covalentlylinked, with the proviso that the covalent link is not a peptide bond;wherein the first domain and the fifth domain associate to form a firstbinding site (VL1)(VH1) that binds the first epitope; wherein the seconddomain and the fourth domain associate to form a second binding site(VL2)(VH2) that binds the second epitope.

In certain embodiments, the present invention is directed to a covalentbispecific diabody, which diabody is a dimer of diabody molecules, eachdiabody molecule comprising a first and a second polypeptide chain,which first polypeptide chain comprises (i) a first domain comprising abinding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; and whichsecond polypeptide chain comprises (i) a fourth domain comprising abinding region of a light chain variable domain of the secondimmunoglobulin (VL2), (ii) a fifth domain comprising a binding region ofa heavy chain variable domain of the first immunoglobulin (VH1), and(iii) a sixth domain comprising at least one cysteine residue, whichfourth and fifth domains are covalently linked such that the fourth andfifth domains do not associate to form an epitope binding site; andwherein the first polypeptide chain and the second polypeptide chain ofeach diabody molecule are covalently linked, with the proviso that thecovalent link is not a peptide bond; wherein the first domain and thefifth domain of each diabody molecule associate to form a first bindingsite (VL1)(VH1) that binds the first epitope; wherein the second domainand the fourth domain of each diabody molecule associate to form asecond binding site (VL2)(VH2) that binds the second epitope.

In yet other embodiments, the present invention is directed to acovalent tetrapecific diabody, which diabody is a dimer of diabodymolecules, the first diabody molecule comprising a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope and (iii) athird domain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; and whichsecond polypeptide chain comprises (i) a fourth domain comprising abinding region of a light chain variable domain of the secondimmunoglobulin (VL2), (ii) a fifth domain comprising a binding region ofa heavy chain variable domain of the first immunoglobulin (VH1), and(iii) a sixth domain comprising at least one cysteine residue, whichfourth and fifth domains are covalently linked such that the fourth andfifth domains do not associate to form an epitope binding site; andwherein the first polypeptide chain and the second polypeptide chain arecovalently linked, with the proviso that the covalent link is not apeptide bond; wherein the first domain and the fifth domain associate toform a first binding site (VL1)(VH1) that binds the first epitope;wherein the second domain and the fourth domain associate to form asecond binding site (VL2)(VH2) that binds the second epitope; and thesecond diabody molecule comprising a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a thirdimmunoglobulin (VL3) specific for a third epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a fourthimmunoglobulin (VH4) specific for a fourth epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; and whichsecond polypeptide chain comprises (i) a fourth domain comprising abinding region of a light chain variable domain of the fourthimmunoglobulin (VL4), (ii) a fifth domain comprising a binding region ofa heavy chain variable domain of the third immunoglobulin (VH3), and(iii) a sixth domain comprising at least one cysteine residue, whichfourth and fifth domains are covalently linked such that the fourth andfifth domains do not associate to form an epitope binding site; andwherein the first polypeptide chain and the second polypeptide chain arecovalently linked, with the proviso that the covalent link is not apeptide bond; wherein the first domain and the fifth domain associate toform a first binding site (VL3)(VH3) that binds the third epitope;wherein the second domain and the fourth domain associate to form asecond binding site (VL4)(VH4) that binds the fourth epitope.

In certain aspects of the invention the first epitope, second epitope,and where applicable, third epitope and fourth epitope can be the same.In other aspects, the first epitope, second epitope, and whereapplicable, third epitope and fourth epitope can each different from theother. In certain aspects of the invention comprising a third epitopebinding domain, the first epitope and third epitope can be the same. Incertain aspects of the invention comprising a fourth epitope bindingdomain, the first epitope and fourth epitope can be the same. In certainaspects of the invention comprising a third epitope binding domain, thesecond epitope and third epitope can be the same. In certain aspects ofthe invention comprising a fourth epitope binding domain, the secondepitope and fourth epitope can be the same. In preferred aspects of theinvention, the first epitope and second epitope are different. In yetother aspects of the invention comprising a third epitope binding domainand a fourth epitope binding domain, the third epitope and fourthepitope can be different. It is to be understood that any combination ofthe foregoing is encompassed in the present invention.

In particular aspects of the invention, the first domain and the fifthdomain of the diabody or diabody molecule can be derived from the sameimmunoglobulin. In another aspect, the second domain and the fourthdomain of the diabody or diabody molecule can be derived from the sameimmunoglobulin. In yet another aspect, the first domain and the fifthdomain of the diabody or diabody molecule can be derived from adifferent immunoglobulin. In yet another aspect, the second domain andthe fourth domain of the diabody or diabody molecule can be derived froma different immunoglobulin. It is to be understood that any combinationof the foregoing is encompassed in the present invention.

In certain aspects of the invention, the covalent linkage between thefirst polypeptide chain and second polypeptide chain of the diabody ordiabody molecule can be via a disulfide bond between at least onecysteine residue on the first polypeptide chain and at least onecysteine residue on the second polypeptide chain. The cysteine residueson the first or second polypeptide chains that are responsible fordisulfide bonding can be found anywhere on the polypeptide chainincluding within the first, second, third, fourth, fifth and sixthdomains. In a specific embodiment the cysteine residue on the firstpolypeptide chain is found in the first domain and the cysteine residueon the second polypeptide chain is found in the fifth domain. The first,second, fourth and fifth domains correspond to the variable regionsresponsible for binding. In preferred embodiments, the cysteine residuesresponsible for the disulfide bonding between the first and secondpolypeptide chains are located within the third and sixth domains,respectively. In a particular aspect of this embodiment, the thirddomain of the first polypeptide chain comprises the C-terminal 6 aminoacids of the human kappa light chain, FNRGEC (SEQ ID NO: 23), which canbe encoded by the amino acid sequence (SEQ ID NO: 17). In another aspectof this embodiment, the sixth domain of the second polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO: 23), which can be encoded by the amino acid sequence(SEQ ID NO: 17). In still another aspect of this embodiment, the thirddomain of the first polypeptide chain comprises the amino acid sequenceVEPKSC (SEQ ID NO: 77), derived from the hinge domain of a human IgG,and which can be encoded by the nucleotide sequence (SEQ ID NO: 78). Inanother aspect of this embodiment, the sixth domain of the secondpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:77), derived from the hinge domain of a human IgG, and which can beencoded by the nucleotide sequence (SEQ ID NO: 78). In certain aspectsof this embodiment, the third domain of the first polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO: 23); and the sixth domain of the second polypeptidechain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77). In otheraspects of this embodiment, the sixth domain of the second polypeptidechain comprises the C-terminal 6 amino acids of the human kappa lightchain, FNRGEC (SEQ ID NO: 23); and the third domain of the firstpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:77). In yet other aspects of this embodiment, the third domain of thefirst polypeptide chain comprises the C-terminal 6 amino acids of thehuman kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain ofthe second polypeptide chain comprises a hinge domain. In other aspectsof this embodiment, the sixth domain of the second polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptidechain comprises the hinge domain. In yet other aspects of thisembodiment, the third domain of the first polypeptide chain comprisesthe C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQID NO: 23); and the sixth domain of the first polypeptide chaincomprises an Fc domain, or portion thereof. In still other aspects ofthis embodiment, the sixth domain of the second polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptidechain comprises an Fc domain, or portion thereof.

In other embodiments, the cysteine residues on the first or secondpolypeptide that are responsible for the disulfide bonding can belocated outside of the first, second or third domains on the firstpolypeptide chain and outside of the fourth, fifth and sixth domain onthe second polypeptide chain. In particular, the cysteine residue on thefirst polypeptide chain can be N-terminal to the first domain or can beC-terminal to the first domain. The cysteine residue on the firstpolypeptide chain can be N-terminal to the second domain or can beC-terminal to the second domain. The cysteine residue on the firstpolypeptide chain can be N-terminal to the third domain or can beC-terminal to the third domain. Further, the cysteine residue on thesecond polypeptide chain can be N-terminal to the fourth domain or canbe C-terminal to the fourth domain. The cysteine residue on the secondpolypeptide chain can be N-terminal to the fifth domain or can beC-terminal to the fifth domain. Accordingly, the cysteine residue on thesecond polypeptide chain can be C-terminal to the sixth domain or can beN-terminal to the sixth domain. In a particular aspect, disulfide bondcan between at least two cysteine residues on the first polypeptidechain and at least two cysteine residues on the second polypeptidechain. In a particular aspect, wherein the third domain and sixth domaindo not comprise an Fc domain, or portion thereof, the cysteine residuecan be at the C-terminus of the first polypeptide chain and at theC-terminus of the second polypeptide chain. It is to be understood thatany combination of the foregoing is encompassed in the presentinvention.

In specific embodiments of the invention described supra, the covalentdiabody of the invention encompasses dimers of diabody molecules,wherein each diabody molecule comprises a first and second polypeptidechain. In certain aspects of this embodiment the diabody molecules canbe covalently linked to form the dimer, with the proviso that thecovalent linkage is not a peptide bond. In preferred aspects of thisembodiment, the covalent linkage is a disulfide bond between at leastone cysteine residue on the first polypeptide chain of each of thediabody molecules of the dimer. In yet more preferred aspects of thisinvention, the covalent linkage is a disulfide bond between at least onecysteine residue on the first polypeptide chain of each of the diabodymolecules forming the dimer, wherein said at least one cysteine residueis located in the third domain of each first polypeptide chain.

In certain aspects of the invention, the first domain on the firstpolypeptide chain can be N-terminal to the second domain or can beC-terminal to the second domain. The first domain on the firstpolypeptide chain can be N-terminal to the third domain or can beC-terminal to the third domain. The second domain on the firstpolypeptide chain can be N-terminal to the first domain or can beC-terminal to the first domain. Further, the second domain on the firstpolypeptide chain can be N-terminal to the third domain or can beC-terminal to the third domain. Accordingly, the third domain on thefirst polypeptide chain can be N-terminal to the first domain or can beC-terminal to the first domain. The third domain on the firstpolypeptide chain can be N-terminal to the second domain or can beC-terminal to the second domain. With respect to the second polypeptidechain, the fourth domain can be N-terminal to the fifth domain or can beC-terminal to the fifth domain. The fourth domain can be N-terminal tothe sixth domain or can be C-terminal to the sixth domain. The fifthdomain on the second polypeptide chain can be N-terminal to the fourthdomain or can be C-terminal to the fourth domain. The fifth domain onthe second polypeptide chain can be N-terminal to the sixth domain orcan be C-terminal to the sixth domain. Accordingly the sixth domain onthe second polypeptide chain can be N-terminal to the fourth domain orcan be C-terminal to the fourth domain. The sixth domain on the secondpolypeptide chain can be N-terminal to the fifth domain or can beC-terminal to the fifth domain. It is to be understood that anycombination of the foregoing is encompassed in the present invention.

In certain embodiments, first domain and second domain can be locatedC-terminal to the third domain on the first polypeptide chain; or thefirst domain and second domain can be located N-terminal to the thirddomain on the first polypeptide chain. With respect to the secondpolypeptide chain, the fourth domain and fifth domain can be locatedC-terminal to the sixth domain, or the fourth domain and fifth domaincan be located N-terminal to the sixth domain. In certain aspects ofthis embodiment, the present invention is directed to a covalentbispecific diabody, which diabody is a dimer of diabody molecules, eachdiabody molecule comprising a first and a second polypeptide chain,which first polypeptide chain comprises (i) a first domain comprising abinding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site and wherein thethird domain is located N-terminal to both the first domain and seconddomain; and which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and (iii) a sixth domain comprising at least one cysteineresidue, which fourth and fifth domains are covalently linked such thatthe fourth and fifth domains do not associate to form an epitope bindingsite; and wherein the first polypeptide chain and the second polypeptidechain of each diabody molecule are covalently linked, with the provisothat the covalent link is not a peptide bond; wherein the first domainand the fifth domain of each diabody molecule associate to form a firstbinding site (VL1)(VH1) that binds the first epitope; wherein the seconddomain and the fourth domain of each diabody molecule associate to forma second binding site (VL2)(VH2) that binds the second epitope.

In yet another embodiment, the present invention is directed to acovalent tetrapecific diabody, which diabody is a dimer of diabodymolecules, the first diabody molecule comprising a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope and (iii) athird domain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site and wherein thethird domain is located N-terminal to both the first domain and seconddomain; and which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and (iii) a sixth domain comprising at least one cysteineresidue, which fourth and fifth domains are covalently linked such thatthe fourth and fifth domains do not associate to form an epitope bindingsite; and wherein the first polypeptide chain and the second polypeptidechain are covalently linked, with the proviso that the covalent link isnot a peptide bond; wherein the first domain and the fifth domainassociate to form a first binding site (VL1)(VH1) that binds the firstepitope; wherein the second domain and the fourth domain associate toform a second binding site (VL2)(VH2) that binds the second epitope; andthe second diabody molecule comprises a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a thirdimmunoglobulin (VL3) specific for a third epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a fourthimmunoglobulin (VH4) specific for a fourth epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site and wherein thethird domain is located N-terminal to both the first domain and seconddomain; and which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thefourth immunoglobulin (VL4), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the third immunoglobulin(VH3), and (iii) a sixth domain comprising at least one cysteineresidue, which fourth and fifth domains are covalently linked such thatthe fourth and fifth domains do not associate to form an epitope bindingsite; and wherein the first polypeptide chain and the second polypeptidechain are covalently linked, with the proviso that the covalent link isnot a peptide bond; wherein the first domain and the fifth domainassociate to form a first binding site (VL3)(VH3) that binds the thirdepitope; wherein the second domain and the fourth domain associate toform a second binding site (VL4)(VH4) that binds the fourth epitope.

As discussed above, the domains on the individual polypeptide chains arecovalently linked. In specific aspects, the covalent link between thefirst and second domain, first and third domain, second and thirddomain, fourth and fifth domain, fourth and sixth domain, and/or fifthand sixth domain can be a peptide bond. In particular, the first andsecond domains, and the fourth and fifth domains can be separated by thethird domain and sixth domain, respectively, or by additional amino acidresidues, so long as the first and second, and fourth and fifth domainsdo not associate to form a binding site. The number of amino acidresidues can be 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues. Inone preferred aspect, the number of amino acid residues between thedomains is 8.

In certain aspects of the invention, the domains of the first and secondpolypeptide chain comprising an Fc domain, i.e., optionally, the thirdand sixth domains, respectively, can further comprise a hinge domainsuch that the domain comprises a hinge-Fc region. In alternativeembodiments, the first polypeptide chain or the second polypeptide chaincan comprise a hinge domain without also comprising an Fc domain. Theheavy chains, light chains, hinge regions, Fc domains, and/or hinge-Fcdomains for use in the invention can be derived from any immunoglobulintype including IgA, IgD, IgE, IgG or IgM. In a preferred aspect, theimmunoglobulin type is IgG, or any subtype thereof, i.e., IgG₁, IgG₂,IgG₃ or IgG₄. In other aspects, the immunoglobulin from which the lightand heavy chains are derived is humanized or chimerized.

Further, the first epitope and second epitopes, and, where applicable,third epitope and fourth epitope, to which the diabody or diabodymolecule binds can be different epitopes from the same antigen or can bedifferent epitopes from different antigens. The antigens can be anymolecule to which an antibody can be generated. For example, proteins,nucleic acids, bacterial toxins, cell surface markers, autoimmunemarkers, viral proteins, drugs, etc. In particular aspects, at least oneepitope binding site of the diabody is specific for an antigen on aparticular cell, such as a B-cell, a T-cell, a phagocytic cell, anatural killer (NK) cell or a dendritic cell.

In certain aspects of the present embodiment, at least one epitopebinding site of the diabody or diabody molecule is specific for a Fcreceptor, which Fc receptor can be an activating Fc receptor or aninhibitory Fc receptor. In particular aspects, the Fc receptor is a Fcγreceptor, and the Fcγ receptor is a FcγRI, FcγRII or FcγRIII receptor.In more preferred aspects, the FcγRIII receptor is the FcγRIIIA (CD16A)receptor or the FcγRIIIB (CD 16B) receptor, and, more preferably, theFcγRIII receptor is the FcγRIIIA (CD16A) receptor. In another preferredaspect, the FcγRII receptor is the FcγRIIA (CD32A) receptor or theFcγRIIB (CD32B) receptor, and more preferably the FcγRIIB (CD32B)receptor. In a particularly preferred aspect, one binding site of thediabody is specific for CD32B and the other binding site is specific forCD16A. In a specific embodiment of the invention, at least one epitopebinding site of the diabody or diabody molecule is specific for anactivating Fc receptor and at least one other site is specific for aninhibitory Fc receptor. In certain aspects of this embodiment theactivating Fc receptor is CD32A and the inhibitory Fc receptor is CD32B.In other aspects of this embodiment the activating Fc receptor is BCRand the inhibitory Fc receptor is CD32B. In still other aspects of thisembodiment, the activating Fc receptor is IgER1 and the inhibitory Fcreceptor is CD32B.

In cases where one epitope binding site is specific for CD16A, the VLand VH domains can be the same as or similar to the VL and VH domains ofthe mouse antibody 3G8, the sequence of which has been cloned and is setforth herein. In other cases where one epitope binding site is specificfor CD32A, the VL and VH domains can be the same as or similar to the VLand VH domains of the mouse antibody IV.3. In yet other cases where oneepitope binding site is specific for CD32B, the VL and VH domains can bethe same as or similar to the VL and VH domains of the mouse antibody2B6, the sequence of which has been cloned and is set forth herein. Itis to be understood that any of the VL or VH domains of the 3G8, 2B6 andIV.3 antibodies can be used in any combination. The present invention isalso directed to a bispecific diabody or diabody molecule wherein thefirst epitope is specific for CD32B, and the second epitope is specificfor CD16A.

In other aspects, an epitope binding site can be specific for apathogenic antigen. As used herein, a pathogenic antigen is an antigeninvolved in a specific pathogenic disease, including cancer, infectionand autoimmune disease. Thus, the pathogenic antigen can be a tumorantigen, a bacterial antigen, a viral antigen, or an autoimmune antigen.Exemplary pathogenic antigens include, but are not limited tolipopolysaccharide, viral antigens selected from the group consisting ofviral antigens from human immunodeficiency virus, Adenovirus,Respiratory Syncitial Virus, West Nile Virus (e.g., E16 and/or E53antigens) and hepatitis virus, nucleic acids (DNA and RNA) and collagen.Preferably, the pathogenic antigen is a neutralizing antigen. In apreferred aspect, where one epitope binding site is specific for CD16Aor CD32A, the other epitope binding site is specific for a pathogenicantigen excluding autoimmune antigens. In yet another preferred aspect,where one epitope binding site is specific for CD32B, the other epitopebinding site is specific for any pathogenic antigen. In specificembodiments, the diabody molecule of the invention binds two differentantigens on the same cell, for example, one antigen binding site isspecific for an activating Fc receptor while the other is specific foran inhibitory Fc receptor. In other embodiments, the diabody moleculebinds two distinct viral neutralizing epitopes, for example, but notlimited to, E16 and E53 of West Nile Virus.

In yet another embodiment of the present invention, the diabodies of theinvention can be used to treat a variety of diseases and disorders.Accordingly, the present invention is directed to a method for treatinga disease or disorder comprising administering to a patient in needthereof an effective amount of a covalent diabody or diabody molecule ofthe invention in which at least one binding site is specific for apathogenic antigen, such as an antigen expressed on the surface of acancer cell or on the surface of a bacterium or virion and at least oneother binding site is specific for a Fc receptor, e.g., CD16A.

In yet another embodiment, the invention is directed to a method fortreating a disease or disorder comprising administering to a patient inneed thereof an effective amount of a diabody or diabody molecule of theinvention, in which at least one binding site is specific for CD32B andat least one other binding site is specific for CD16A.

In yet another embodiment, the invention is directed to a method forinducing immune tolerance to a pathogenic antigen comprisingadministering to a patient in need there an effective amount of acovalent diabody or covalent diabody molecule of the invention, in whichat least one binding site is specific for CD32B and at least one otherbinding site is specific for said pathogenic antigen. In aspects of thisembodiment, the pathogenic antigen can be an allergen or anothermolecule to which immune tolerance is desired, such as a proteinexpressed on transplanted tissue.

In yet another embodiment, the present invention is directed to a methodfor detoxification comprising administering to a patient in need thereofan effective amount of a covalent diabody or diabody molecule of theinvention, in which at least one binding site is specific for a cellsurface marker and at least one other binding site is specific for atoxin. In particular aspects, the diabody of the invention administeredis one where one binding site is specific for a cell surface marker suchas an Fc and the other binding site is specific for a bacterial toxin orfor a drug. In one aspect, the cell surface marker is not found on redblood cells.

3.1 Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. The practice of the present invention willemploy, unless otherwise indicated, conventional techniques of molecularbiology (including recombinant techniques), microbiology, cell biology,biochemistry, nucleic acid chemistry, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Current Protocols in Immunology (J. E. Coligan etal., eds., 1999, including supplements through 2001); Current Protocolsin Molecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); Molecular Cloning: A Laboratory Manual, thirdedition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); The Immunoassay Handbook (D. Wild, ed.,Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson,ed., Academic Press, 1996); Methods of Immunological Analysis (R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlagsgesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1999; and Beaucage et al. eds., Current Protocols in Nucleic AcidChemistry John Wiley & Sons, Inc., New York, 2000).

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” “specificallyrecognizes” and analogous terms refer to molecules that specificallybind to an antigen (e.g., eptiope or immune complex) and do notspecifically bind to another molecule. A molecule that specificallybinds to an antigen may bind to other peptides or polypeptides withlower affinity as determined by, e.g., immunoassays, BIAcore, or otherassays known in the art. Preferably, molecules that specifically bind anantigen do not cross-react with other proteins. Molecules thatspecifically bind an antigen can be identified, for example, byimmunoassays, BIAcore, or other techniques known to those of skill inthe art.

As used herein, “immune complex” refers to a structure which forms whenat least one target molecule and at least one heterologous Feyregion-containing polypeptide bind to one another forming a largermolecular weight complex. Examples of immune complexes areantigen-antibody complexes which can be either soluble or particulate(e.g., an antigen/antibody complex on a cell surface.).

As used herein, the terms “heavy chain,” “light chain,” “variableregion,” “framework region,” “constant domain,” and the like, have theirordinary meaning in the immunology art and refer to domains in naturallyoccurring immunoglobulins and the corresponding domains of synthetic(e.g., recombinant) binding proteins (e.g., humanized antibodies, singlechain antibodies, chimeric antibodies, etc.). The basic structural unitof naturally occurring immunoglobulins (e.g., IgG) is a tetramer havingtwo light chains and two heavy chains, usually expressed as aglycoprotein of about 150,000 Da. The amino-terminal (“N”) portion ofeach chain includes a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. Thecarboxy-terminal (“C”) portion of each chain defines a constant region,with light chains having a single constant domain and heavy chainsusually having three constant domains and a hinge region. Thus, thestructure of the light chains of an IgG molecule is n-V_(L)-C_(L)-c andthe structure of IgG heavy chains is n-V_(H)-C_(H1)-H-C_(H2)-C_(H3)-c(where H is the hinge region). The variable regions of an IgG moleculeconsist of the complementarity determining regions (CDRs), which containthe residues in contact with antigen and non-CDR segments, referred toas framework segments, which in general maintain the structure anddetermine the positioning of the CDR loops (although certain frameworkresidues may also contact antigen). Thus, the V_(L) and V_(H) domainshave the structure n-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4-c.

When referring to binding proteins or antibodies (as broadly definedherein), the assignment of amino acids to each domain is in accordancewith the definitions of Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1987 and 1991).Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated by the position of an aminoacid in the chain. Kabat described numerous amino acid sequences forantibodies, identified an amino acid consensus sequence for eachsubgroup, and assigned a residue number to each amino acid. Kabat'snumbering scheme is extendible to antibodies not included in hiscompendium by aligning the antibody in question with one of theconsensus sequences in Kabat by reference to conserved amino acids. Thismethod for assigning residue numbers has become standard in the fieldand readily identifies amino acids at equivalent positions in differentantibodies, including chimeric or humanized variants. For example, anamino acid at position 50 of a human antibody light chain occupies theequivalent position to an amino acid at position 50 of a mouse antibodylight chain.

As used herein, the term “heavy chain” is used to define the heavy chainof an IgG antibody. In an intact, native IgG, the heavy chain comprisesthe immunoglobulin domains VH, CH1, hinge, CH2 and CH3. Throughout thepresent specification, the numbering of the residues in an IgG heavychain is that of the EU index as in Kabat et al., Sequences of Proteinsof Immunological Interest, 5^(th) Ed. Public Health Service, NH1, MD(1991), expressly incorporated herein by references. The “EU index as inKabat” refers to the numbering of the human IgG1 EU antibody. Examplesof the amino acid sequences containing human IgG1 hinge, CH2 and CH3domains are shown in FIGS. 1A and 1B as described, infra. FIGS. 1A and1B also set forth amino acid sequences of the hinge, CH2 and CH3 domainsof the heavy chains of IgG2, IgG3 and IgG4. The amino acid sequences ofIgG2, IgG3 and IgG4 isotypes are aligned with the IgG1 sequence byplacing the first and last cysteine residues of the respective hingeregions, which form the inter-heavy chain S—S bonds, in the samepositions. For the IgG2 and IgG3 hinge region, not all residues arenumbered by the EU index.

The “hinge region” or “hinge domain” is generally defined as stretchingfrom Glu216 to Pro230 of human IgG 1. An example of the amino acidsequence of the human IgG1 hinge region is shown in FIG. 1A (amino acidresidues in FIG. 1A are numbered according to the Kabat system). Hingeregions of other IgG isotypes may be aligned with the IgG1 sequence byplacing the first and last cysteine residues forming inter-heavy chainS—S binds in the same positions as shown in FIG. 1A.

As used herein, the term “Fc region,” “Fc domain” or analogous terms areused to define a C-terminal region of an IgG heavy chain. An example ofthe amino acid sequence containing the human IgG1 is shown in FIG. 1B.Although boundaries may vary slightly, as numbered according to theKabat system, the Fc domain extends from amino acid 231 to amino acid447 (amino acid residues in FIG. 1B are numbered according to the Kabatsystem). FIG. 1B also provides examples of the amino acid sequences ofthe Fc regions of IgG isotypes IgG2, IgG3, and IgG4.

The Fc region of an IgG comprises two constant domains, CH2 and CH3. TheCH2 domain of a human IgG Fc region usually extends from amino acids 231to amino acid 341 according to the numbering system of Kabat (FIG. 1B).The CH3 domain of a human IgG Fc region usually extends from amino acids342 to 447 according to the numbering system of Kabat (FIG. 1B). The CH2domain of a human IgG Fc region (also referred to as “Cγ2” domain) isunique in that it is not closely paired with another domain. Rather, twoN-linked branched carbohydrate chains are interposed between the two CH2domains of an intact native IgG.

As used herein the terms “FcγR binding protein,” “FcγR antibody,” and“anti-FcγR antibody”, are used interchangeably and refer to a variety ofimmunoglobulin-like or immunoglobulin-derived proteins. “FcγR bindingproteins” bind FcγR via an interaction with V_(L) and/or V_(H) domains(as distinct from Fcγ-mediated binding). Examples of FcγR bindingproteins include fully human, polyclonal, chimeric and humanizedantibodies (e.g., comprising 2 heavy and 2 light chains), fragmentsthereof (e.g., Fab, Fab′, F(ab′)₂, and Fv fragments), bifunctional ormultifunctional antibodies (see, e.g., Lanzavecchia et al. (1987) “TheUse Of Hybrid Hybridomas To Target Human Cytotoxic T Lymphocytes,” Eur.J. Immunol. 17:105-111), single chain antibodies (see, e.g., Bird et al.(1988) “Single-Chain Antigen-Binding Proteins,” Science 242:423-26),fusion proteins (e.g., phage display fusion proteins), “minibodies”(see, e.g., U.S. Pat. No. 5,837,821) and other antigen binding proteinscomprising a V_(L) and/or V_(H) domain or fragment thereof. In oneaspect, the FcγRIIIA binding protein is a “tetrameric antibody” i.e.,having generally the structure of a naturally occurring IgG andcomprising variable and constant domains, i.e., two light chainscomprising a V_(L) domain and a light chain constant domain and twoheavy chains comprising a V_(H) domain and a heavy chain hinge andconstant domains.

As used herein the term “FcγR antagonists” and analogous terms refer toprotein and non-proteinacious substances, including small moleculeswhich antagonize at least one biological activity of an FcγR, e.g.,block signaling. For example, the molecules of the invention blocksignaling by blocking the binding of IgGs to an FcγR.

As used herein, the term “derivative” in the context of polypeptides orproteins refers to a polypeptide or protein that comprises an amino acidsequence which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative” asused herein also refers to a polypeptide or protein which has beenmodified, i.e, by the covalent attachment of any type of molecule to thepolypeptide or protein. For example, but not by way of limitation, anantibody may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularan antigen or other protein, etc. A derivative polypeptide or proteinmay be produced by chemical modifications using techniques known tothose of skill in the art, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Further, a derivative polypeptide or proteinderivative possesses a similar or identical function as the polypeptideor protein from which it was derived.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated.

As used herein, the term “diabody molecule” refers to a complex of twoor more polypeptide chains or proteins, each comprising at least one VLand one VH domain or fragment thereof, wherein both domains arecomprised within a single polypeptide chain. In certain embodiments“diabody molecule” includes molecules comprising an Fc or a hinge-Fcdomain. Said polypeptide chains in the complex may be the same ordifferent, i.e., the diabody molecule may be a homo-multimer or ahetero-multimer. In specific aspects, “diabody molecule” includes dimersor tetramers or said polypeptide chains containing both a VL and VHdomain. The individual polypeptide chains comprising the multimericproteins may be covalently joined to at least one other peptide of themultimer by interchain disulfide bonds.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

“Identical polypeptide chains” as used herein also refers to polypeptidechains having almost identical amino acid sequence, for example,including chains having one or more amino acid differences, preferablyconservative amino acid substitutions, such that the activity of the twopolypeptide chains is not significantly different

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. In someembodiments, cancer refers to a benign tumor, which has remainedlocalized. In other embodiments, cancer refers to a malignant tumor,which has invaded and destroyed neighboring body structures and spreadto distant sites. In some embodiments, the cancer is associated with aspecific cancer antigen.

As used herein, the term “immunomodulatory agent” and variations thereofrefer to an agent that modulates a host's immune system. In certainembodiments, an immunomodulatory agent is an immunosuppressant agent. Incertain other embodiments, an immunomodulatory agent is animmunostimulatory agent. Immunomodatory agents include, but are notlimited to, small molecules, peptides, polypeptides, fusion proteins,antibodies, inorganic molecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to a fragment of a polypeptideor protein or a non-protein molecule having antigenic or immunogenicactivity in an animal, preferably in a mammal, and most preferably in ahuman. An epitope having immunogenic activity is a fragment of apolypeptide or protein that elicits an antibody response in an animal.An epitope having antigenic activity is a fragment of a polypeptide orprotein to which an antibody immunospecifically binds as determined byany method well-known to one of skill in the art, for example byimmunoassays. Antigenic epitopes need not necessarily be immunogenic.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder. A therapeutically effective amount may refer to the amountof therapeutic agent sufficient to delay or minimize the onset ofdisease, e.g., delay or minimize the spread of cancer. A therapeuticallyeffective amount may also refer to the amount of the therapeutic agentthat provides a therapeutic benefit in the treatment or management of adisease. Further, a therapeutically effective amount with respect to atherapeutic agent of the invention means the amount of therapeutic agentalone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of a disease.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of one or more symptoms ofa disorder in a subject as result of the administration of aprophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder. Afirst prophylactic or therapeutic agent can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent to asubject with a disorder.

“Effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or an antigen. Effector functions include but are not limitedto antibody dependent cell mediated cytotoxicity (ADCC), antibodydependent cell mediated phagocytosis (ADCP), and complement dependentcytotoxicity (CDC). Effector functions include both those that operateafter the binding of an antigen and those that operate independent ofantigen binding.

“Effector cell” as used herein is meant a cell of the immune system thatexpresses one or more Fc receptors and mediates one or more effectorfunctions. Effector cells include but are not limited to monocytes,macrophages, neutrophils, dendritic cells, eosinophils, mast cells,platelets, B cells, large granular lymphocytes, Langerhans' cells,natural killer (NK) cells, and may be from any organism including butnot limited to humans, mice, rats, rabbits, and monkeys.

As used herein, the term “specifically binds an immune complex” andanalogous terms refer to molecules that specifically bind to an immunecomplex and do not specifically bind to another molecule. A moleculethat specifically binds to an immune complex may bind to other peptidesor polypeptides with lower affinity as determined by, e.g.,immunoassays, BIAcore, or other assays known in the art. Preferably,molecules that specifically bind an immune complex do not cross-reactwith other proteins. Molecules that specifically bind an immune complexcan be identified, for example, by immunoassays, BIAcore, or othertechniques known to those of skill in the art.

A “stable fusion protein” as used herein refers to a fusion protein thatundergoes minimal to no detectable level of degradation duringproduction and/or storage as assessed using common biochemical andfunctional assays known to one skilled in the art, and can be stored foran extended period of time with no loss in biological activity, e.g.,binding to FcγR.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B Amino Acid Sequence of Human IgG CH1, Hinge and Fc Regions

FIG. 1 provides the amino acid sequences of human IgG1, IgG2, IgG3 andIgG4 hinge (A) and Fc (B) domains. (IgG1 hinge domain (SEQ ID NO: 1);IgG2 hinge domain (SEQ ID NO: 2); IgG3 hinge domain (SEQ ID NO: 3); IgG4hinge domain (SEQ ID NO: 4); IgG1 Fc domain (SEQ ID NO: 5); IgG2 Fcdomain (SEQ ID NO: 6); IgG3 Fc domain (SEQ ID NO: 7); IgG1 Fc domain(SEQ ID NO: 8)). The amino acid residues shown in FIGS. 1A and 1B arenumbered according to the numbering system of Kabat EU. Isotypesequences are aligned with the IgG1 sequence by placing the first andlast cysteine residues of the respective hinge regions, which form theinter-heavy chain S—S bonds, in the same positions. For FIG. 1B,residues in the CH2 domain are indicated by +, while residues in the CH3domain are indicated by −.

FIG. 2 Schematic Representation of Polypeptide Chains of CovalentBifunctional Diabodies

Polypeptides of a covalent, bifunctional diabody consist of an antibodyVL and an antibody VH domain separated by a short peptide linker. The 8amino acid residue linker prevents self assembly of a single polypeptidechain into scFv constructs, and, instead, interactions between the VLand VH domains of differing polypeptide chains predominate. 4 constructswere created (each construct is described from the amino terminus (“n”),left side of the construct, to the carboxy terminus (“c”), right side offigure): construct (1) (SEQ ID NO: 9) comprised, n—the VL domainHu2B6—linker (GGGSGGGG (SEQ ID NO: 10))—the VH domain of Hu3G8—and aC-terminal sequence (LGGC)-c; construct (2) (SEQ ID NO: 11) comprisedn—the VL domain Hu3G8—linker (GGGSGGGG (SEQ ID NO: 10))—the VH domain ofHu2B6—and a C-terminal sequence (LGGC)-c; construct (3) (SEQ ID NO: 12)comprised n—the VL domain Hu3G8—linker (GGGSGGGG (SEQ ID NO: 10))—the VHdomain of Hu3G8—and a C-terminal sequence (LGGC)-c; construct (4) (SEQID NO: 13) comprised n—the VL domain Hu2B6—linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu2B6—and a C-terminal sequence (LGGC)-c.

FIG. 3 SDS-PAGE Analysis of Affinity Purified Diabodies

Affinity purified diabodies were subjected to SDS-PAGE analysis underreducing (lanes 1-3) or non-reducing (lanes 4-6) conditions. Approximatemolecular weights of the standard (in between lanes 3 and 4) areindicated. Lanes 1 and 4, h3G8 CMD; Lanes 2 and 5, h2B6 CMD; and Lanes 3and 6, h2B6-h3G8 CBD.

FIGS. 4 A-B SEC Analysis of Affinity Purified Diabodies

Affinity purified diabodies were subjected to SEC analysis. (A) Elutionprofile of known standards: full-length IgG (˜150 kDa), Fab fragment ofIgG (˜50 kDa), and scFv (˜30 kDa); (B) Elution profile of h2b6 CMD, h3G8CMD, and h2B6-h3G8 CBD.

FIG. 5 Binding of h2B6-h3G8 CBD to sCD32B and sCD16A

The binding of h2B6-h3G8 CBD to sCD32B and sCD16A was assayed in asandwich ELISA. sCD32B was used as the target protein. The secondaryprobe was HRP conjugated sCD16A. h3G8 CMD, which binds CD16A, was usedas control.

FIGS. 6 A-C Biacore Analysis of Diabody Binding to sCD16A, sCD32B andsCD32B

The binding of h2B6-h3G8 CBD, h2B6 CMD and h3G8 CMD to sCD16A, sCD32B,and sCD32A (negative control) was assayed by SPR analysis. h3G8 scFv wasalso tested as a control. (A) Binding to sCD16; (B) Binding to sCD32Band (C) Binding to sCD32A. Diabodies were injected at a concentration of100 NM, and scFv at a concentration of 200 nM, over receptor surfaces ata flow rate of 50 ml/min for 60 sec.

FIGS. 7 A-C Biacore Analysis of Diabody Binding to sCD16A and sCD32B

The binding of h2B6-h3G8 CBD, h2B6 CMD and h3G8 CMD to sCD16A, andsCD32B was assayed by SPR analysis. h3G8 scFv was also tested as acontrol. (A) Binding of to h3G8 CMD sCD16A; (B) Binding of h2B6-h3G8 CBDto sCD16A; (C) Binding of h3G8 scFv to sCD16A; (D) Binding of h2B6 CMDto sCD32B; and (E) Binding of h2B6-h3G8 CBD to sCD32B. Diabodies wereinjected at concentrations of 6.25-200 nM over receptor surfaces at aflow rate of 70 ml/min for 180 sec.

FIG. 8 Schematic Depicting the Interaction of Polypeptide ChainsComprising VL and VH Domains to Form a Covalent Bispecific DiabodyMolecule

NH₂ and COOH represent the amino-terminus and carboxy terminus,respectively of each polypeptide chain. S represents the C-terminalcysteine residue on each polypeptide chain. VL and VH indicate thevariable light domain and variable heavy domain, respectively. Dottedand dashed lines are to distinguish between the two polypeptide chainsand, in particular, represent the linker portions of said chains. h2B6Fv and h3G8 Fv indicate an epitope binding site specific for CD32B andCD16, respectively.

FIG. 9 Schematic Representation of Polypeptide Chains Containing FcDomains of Covalent Bispecific Diabodies

Representation of polypeptide constructs of the diabody molecules of theinvention (each construct is described from the amino terminus (“n”),left side of the construct, to the carboxy terminus (“c”), right side offigure). Construct (5) (SEQ ID NO: 14) comprised, n-VL domain Hu2B6—afirst linker (GGGSGGGG (SEQ ID NO: 10))—the VH domain of Hu3G8—a secondlinker (LGGC)—and a C-terminal Fc domain of human IgG1-c; construct (6)(SEQ ID NO: 15) comprised n—the VL domain Hu3G8-linker (GGGSGGGG (SEQ IDNO: 10))—the VH domain of Hu2B6—and second linker (LGGC)—and aC-terminal Fc domain of human IgG1-c; construct (7) (SEQ ID NO: 16)comprised n—the VL domain Hu2B6—a first linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu3G8—and a C-terminal sequence (LGGCFNRGEC) (SEQID NO: 17)-c; construct (8) (SEQ ID NO: 18) comprised n—the VL domainHu3G8—linker (GGGSGGGG (SEQ ID NO: 10))—the VH domain of Hu2B6—andsecond linker (LGGC)—and a C-terminal hinge/Fc domain of human IgG1(with amino acid substitution A215V)-c.

FIG. 10 Binding of Diabody Molecules Comprising Fc Domains to sCD32B andsCD16A

The binding of diabody molecules comprising Fe domains to sCD32B andsCD16A was assayed in a sandwich ELISA. Diabodies assayed were producedby 3 recombinant expression systems: cotransfection of pMGX669 andpMGX674, expressing constructs 1 and 6, respectively; cotransfection ofpMGX667 and pMGX676, expressing constructs 2 and 5, respectively; andcotransfection of pMGX674 and pMGX676, expressing constructs 5 and 6,respectively. sCD32B was used as the target protein. The secondary probewas HRP conjugated sCD16A.

FIG. 11 Schematic Depicting the Interaction of Two Polypeptide Chainseach Comprising an Fc Domain to Form a Bivalent, Covalent Diabody

NH₂ and COOH represent the amino-terminus and carboxy terminus,respectively of each polypeptide chain. S represents the at least onedisulfide bond between a cysteine residue in the second linker sequenceof each polypeptide chain. VL and VH indicate the variable light domainand variable heavy domain, respectively. Dotted and dashed lines are todistinguish between the two polypeptide chains and, in particular,represent the first linker portions of said chains. CH2 and CH3represent the CH2 and CH3 constant domains of an Fc domain. h2B6 Fv andh3G8 Fv indicate an epitope binding site specific for CD32B and CD16,respectively.

FIG. 12 Binding of Diabody Molecules Comprising Hinge/Fc Domains tosCD32B and sCD16A

The binding of diabody molecules comprising Fc domains to sCD32B andsCD16A was assayed in a sandwich ELISA. Diabodies assayed were producedby 4 recombinant expression systems: cotransfection of pMGX669+pMGX674,expressing constructs 1 and 6, respectively; cotransfection ofpMGX669+pMGX678, expressing constructs 2 and 8, respectively;cotransfection of pMGX677+pMGX674, expressing constructs 7 and 6,respectively; and cotransfection of pMGX677+pMGX678, expressingconstructs 7 and 8, respectively. sCD32B was used as the target protein.The secondary probe was HRP conjugated sCD16A.

FIG. 13 Schematic Depicting the Interaction of Polypeptide Chains toForm a Tetrameric Diabody Molecule

NH₂ and COOH represent the amino-terminus and carboxy terminus,respectively of each polypeptide chain. S represents the at least onedisulfide bond between a cysteine residue in the second linker sequencethe Fc bearing, ‘heavier,’ polypeptide chain and a cysteine residue inthe C-terminal sequence of the non-Fc bearing, ‘lighter,’ polypeptidechain. VL and VH indicate the variable light domain and variable heavydomain, respectively. Dotted and dashed lines are to distinguish betweenpolypeptide chains and, in particular, represent the first linkerportions of said heavier chains or the linker of said lighter chains.CH2 and CH3 represent the CH2 and CH3 constant domains of an Fc domain.h2B6 Fv and h3G8 Fv indicate an epitope binding site specific for CD32Band CD16, respectively.

FIG. 14 Schematic Representation of Polypeptides Chains Containing FcDomains which Form Covalent Bispecific Diabodies

Representation of polypeptide constructs which form the diabodymolecules of the invention (each construct is described from the aminoterminus (“n”), left side of the construct, to the carboxy terminus(“c”), right side of figure). Construct (9) (SEQ ID NO: 19) comprisedn—a Hinge/Fc domain of human IgG1—the VL domain Hu3G8—linker (GGGSGGGG(SEQ ID NO: 10))—the VH domain of Hu2B6—linker (GGGSGGGG (SEQ ID NO:10))—and a C-terminal LGGC sequence-c; construct (10) (SEQ ID NO: 20)comprised n—an Fc domain of human IgG 1—the VL domain Hu3G8—linker(GGGSGGGG (SEQ ID NO: 10))—the VH domain of Hu2B6—linker (GGGSGGGG (SEQID NO: 10))—and a C-terminal LGGC sequence-c; construct (11) (SEQ ID NO:21) comprised n—the VL domain Hu2B6 (G105C)—linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu3G8—and a C-terminal hinge/Fc domain of humanIgG1 with amino acid substitution A215V-c; construct (12) (SEQ ID NO:22) comprised n—the VL domain Hu3G8—linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu2B6 (G44C)—and a C-terminal FNRGEC (SEQ ID NO:23) sequence-c.

FIG. 15 A-B SDS-PAGE and Western Blot Analysis of Affinity TetramericDiabodies

Diabodies produced by recombinant expression systems cotransfected withvectors expressing constructs 10 and 1, constructs 9 and 1, andconstructs 11 and 12 were subjected to SDS-PAGE analysis non-reducingconditions (A) and Western Blot analysis using goat anti-human IgG1 H+Las the probe (B). Proteins in the SDS-PAGE gel were visualized withSimply Blue Safestain (Invitrogen). For both panels A and B, diabodymolecules comprising constructs 10 and 1, constructs 9 and 1, andconstructs 11 and 12A are in lanes 1, 2 and 3, respectively.

FIG. 16 Binding of Diabody Molecules Comprising Fc Domains andEngineered Interchain Disulfide Bonds to sCD32B and sCD16A

The binding of diabody molecules comprising Fc domains and engineereddisulfide bonds between the ‘lighter’ and ‘heavier’ polypeptide chainsto sCD32B and sCD16A was assayed in a sandwich ELISA. Diabodies assayedwere produced by 3 recombinant expression systems: expressing constructs1 and 10, expressing constructs 1 and 9, and expressing constructs 11and 12, respectively. sCD32B was used as the target protein. Thesecondary probe was HRP conjugated sCD16A. Binding of h3G8 was used ascontrol.

FIG. 17 Schematic Representation of POLYPROTEIN Precursor of DiabodyMolecule and Schematic Representation of Polypeptide Chains ContainingLambda Light Chain and/or Hinge Domains

Representation of polypeptide constructs which comprise the diabodymolecules of the invention (each construct is described from the aminoterminus (“n”), left side of the construct, to the carboxy terminus(“c”), right side of figure). Construct (13) (SEQ ID NO: 95) comprised,n-VL domain 3G8—a first linker (GGGSGGGG (SEQ ID NO: 10))—the VH domainof 2.4G2VH—a second linker (LGGC)—furin recognition site (RAKR (SEQ IDNO: 93))-VL domain of 2.4G2—a third linker (GGGSGGG (SEQ ID NO: 10)—VHdomain of 3G8—and a C-terminal LGGC domain; (nucleotide sequenceencoding SEQ ID NO: 95 is provided in SEQ ID NO: 96). Construct (14)(SEQ ID NO: 97) comprised, n-VL domain 3G8—a first linker (GGGSGGGG (SEQID NO: 10))—the VH domain of 2.4G2VH—a second linker (LGGC)—furinrecognition site (RAKR (SEQ ID NO: 93))—FMD (Foot and Mouth DiseaseVirus Protease C3) site—VL domain of 2.4G2—a third linker (GGGSGGG (SEQID NO: 10)—VH domain of 3G8—and a C-terminal LGGC domain; (nucleotidesequence encoding SEQ ID NO: 97 is provided in SEQ ID NO: 98). Construct(15) (SEQ ID NO: 99) comprised, n-VL domain Hu2B6—a linker (GGGSGGGG(SEQ ID NO: 10))—the VH domain of Hu3G8—and a C-terminal FNRGEC (SEQ IDNO: 23) domain; (nucleotide sequence encoding SEQ ID NO: 99 is providedin SEQ ID NO: 100). Construct (16) (SEQ ID NO: 101) comprised, n-VLdomain Hu3G8—a linker (GGGSGGGG (SEQ ID NO: 10))—the VH domain ofHu2B6—and a C-terminal VEPKSC (SEQ ID NO: 77) domain; (nucleotidesequence encoding SEQ ID NO: 101 is provided in SEQ ID NO: 102).

FIG. 18 Binding of Diabody Molecules Derived from a PolyproteinPrecursor Molecule to mCD32B and sCD16A

The binding of diabody molecules derived from the polyprotein precursormolecule construct 13 (SEQ ID NO: 95) to murine CD32B (mCD32B) andsoluble CD16A (sCD16A) was assayed in a sandwich ELISA. mCD32B was usedas the target protein. The secondary probe was biotin conjugated sCD16A.

FIG. 19 Binding of Diabody Molecules Comprising Lambda Chain and/orHinge Domains to sCD32B and sCD16A

The binding of diabody molecules comprising domains derived from theC-terminus of the human lambda light chain and/or the hinge domain ofIgG to sCD32B and sCD16A was assayed and compared to the diabodycomprising constructs 1 and 2 (FIG. 5) in a sandwich ELISA. Diabodiesassayed were produced by the recombinant expression system expressingconstructs 15 and 16 (SEQ ID NO: 99 and SEQ ID NO: 101, respectively).sCD32B was used as the target protein. The secondary probe was HRPconjugated sCD16A. Bars with small boxes represent the construct 15/16combination while bars with large boxes represent construct ½combination.

FIG. 20 Schematic Representation of 2B6/4420 DART Bound to CD32B Locatedat the Surface of a Cell and a Fluorescein-Conjugated Molecule

The diagram shows the flexibility of the “universal adaptor”anti-fluorescein arm of the DART as well as the possibility ofsubstituting other specificities for the 2B6 arm. V-regions are shown asboxes, GGGSGGGG (SEQ ID NO: 10) linkers are shown as lines, thedisulfide bond is shown connecting the two chains. The constituents ofone chain are shown in blue while the other is colored pink. N, aminoterminus; C, carboxy terminus; FL, fluorescein, VL, light chain variableregion; VH, heavy chain variable region.

FIG. 21 (Panels A and B) The 2B6/4420 DART Binds Specifically toFluorescein-Conjugated Molecules and can Simultaneously Bind CD32B.

(A) 2B6/4420 or 2B6/3G8 were bound to ELISA plates coated with FITC-SProtein. Binding and function of the 2B6 arm were detected by engagementof soluble CD32B, followed by an antibody specific for CD32B and asecondary detecting antibody conjugated to HRP. (B) 2B6/4420 or 2B6/3G8were bound to ELISA plates coated with HuIgG or FITC-HuIgG(fluorescein-conjugated). Binding was detected by engagement with apolyclonal serum specific for 2B6 Fv followed by an HRP-conjugatedsecondary antibody.

FIG. 22 (Panels A and B) Activation of Purified B Cells Using Anti-HumanCD79B Antibodies.

Purified B cells were activated using increasing concentrations ofanti-human CD79b antibodies FITC-conjugated, CB3.1-FITC (A) orCB3.2-FITC (B) and 50 μg/ml of F(ab′)₂ fragment of GAM IgG Fc specific(x-axis). B cells were activate in the presence of PBS (white bars) or 5μg/ml of either αFITCαCD32BDART (black bars) or αCD16αCD32BDART (greybars). The reactions were performed in triplicate and standarddeviations were calculated.

FIG. 23 (Panels A and B) Activation of Purified B Cells

Purified B cells from a second healthy donor were activated as describedin FIG. 22, Panel B. The proliferation index was measured in cellsactivated in the presence of the anti CD79b antibody FITC-conjugatedCB3.2-FITC (A) and compared to the proliferation index of cellsactivated in the presence of the unlabeled CB3.2 antibody (B).

FIG. 24 (Upper and Lower Panels) In Vivo Mouse B Cell Depletion inhCD16A/B Transgenic Mice Using mGD261

mCD32−/− hCD16A+C57BI/6, mCD32−/− hCD32B+C57BI/6 and mCD32−/−hCD16A+hCD32B+C57BI/6 mice from MacroGenics breeding colony wereinjected IV at days 0, 3, 7, 10, 14 and 17 with MGD261 (10, 3, 1 or 0.3mg/kg), or an irrelevant antibody (hE16 10 mg/kg). Blood was collectedat days—19 (pre-bleed), 4, 11, 18, 25 and 32 for FACS analysis. Animalhealth and activity was recorded three times a week. Upper Panel:h2B6-3G8 and WNV mAb; Lower Panel: h2B6-3G8 hCD16A or hCD32B mice andWNV mAb hCD16A or hCD32B mice.

FIG. 25 In Vivo Mouse B Cell Depletion in hCD16A/B Transgenic Mice Using2.4G2-3G8 DB

mCD16−/−, mCD16−/− hCD16A+C57BI/6, mCD16−/− hCD16B+ and mCD16−/−hCD16A+hCD16B+mice from MacroGenics breeding colony were injected IP atdays 0, 2, 4, 7, 9, 11, 14, 16 and 18 with 2.4G2-3G8 DB (75 ug/mouse),or PBS. Blood was collected at days—10 (pre-bleed), 4, 11 and 18 forFACS analysis. Animal health and activity was recorded three times aweek.

FIG. 26 Demonstration of Anti-Tumor Activity of mGD261 Using anIntravenous (IV) Model of the Human Tumor Cell Line Raji.

Twelve-twenty week old mCD16−/−, hCD16A+, RAG1−/− C57BI/6 mice fromMacroGenics breeding colony were injected IV at day 0 with 5×10⁶ Rajicells. At Days 6, 9, 13, 16, 20, 23, 27 and 30 mice were also treatedintraperitoneously (IP) with 250, 25 or 2.5 ug MGD261 or with PBS(negative control). Mice were then observed daily and body weight wasrecorded twice a week. Mice developing hind leg paralysis weresacrificed.

FIG. 27 DART Expression in a Non-Mammalian Host

BL21DE3 cells (Novagen) were transformed with the pET25b(+)T7-lac+3G8/3G8 plasmid and an amp-resistant colony was used to seedbroth culture. When the culture reached 0.5 OD600 units, 0.5 mM IPTG wasadded to induce expression. The culture was grown at 30° C. for 2 hoursand the cell-free medium was collected.

FIG. 28 DART ELISA

h3G8-h3G8 DART binding ELISA were conducted using 96-well Maxisorpplates. After reaction, the plate was washed with PBS-T three times anddeveloped with 80 μl/well of TMB substrate. After 5 minutes incubation,the reaction was stopped by 40 μl/well of 1% H₂SO₄. The OD450 nm wasread using a 96-well plate reader and SOFTmax software. The read out wasplotted using GraphPadPrism 3.03 software.

FIG. 29 DART-Induced Human B-Cell Death

Human PBMC were incubated overnight with the indicated molecules.Apoptosis was assayed by FACS analysis as the percentage ofPI⁺Annexin-V+ population of B cells (CD20+ cells) on the total FSC/SSCungated population.

FIG. 30 8B5-CB3.1 DART Constructs

Multiple 8B5-CB3.1 DART constructs were produced to illustrate thepresent invention. The construct 5 and 6, or 6 and 7, or 8 and 9, or 9and 10, encoded expression plasmids were co-transfected into HEK-293cells to express 8B5-CB3.1 DART with or without anti flag tag usingLipofectamine 2000 (Invitrogen). The conditioned medium was harvested inevery three days for three times. The conditioned medium was thenpurified using CD32B affinity column.

FIG. 31 8B5-CB3.1 DART ELISA

8B5-CB3.1 DART/ch8B5 competition ELISA were conducted using 96-wellMaxisorp plates. After reaction, the plate was washed with PBS-T threetimes and developed with 80 μl/well of TMB substrate. After 5 minutesincubation, the reaction was stopped by 40 μl/well of 1% H₂SO₄. TheOD450 nm was read using a 96-well plate reader and SOFTmax software. Theread out was plotted using GraphPadPrism 3.03 software.

FIG. 32 Schematic Illustration of Tetravalent DART Structure

Illustrates the general structure of a DART species produced through theassembly of four polypeptide chains. The four antigen-binding domains ofthe Ig-like DART are shown as striped and dark grey ellipses.

FIG. 33 Ig-like Tetravalent DART

Provides a schematic of the epitope binding sites of an Ig-liketetravalent DART.

FIG. 34 mCD32-hCD16A Binding ELISA

Provides the results of ELISAs that demonstrate that the Ig-liketetravalent DART species of Example 6.10 binds antigen with greateraffinity than control (ch-mCD32 mAb) antibody or other DART species.

FIG. 35 Schematic Illustration of Ig DART Molecules

Provides a schematic of Ig DART molecules. Specificity is indicated byshading, pattern or white colored regions, constant regions are shown inblack, and disulfide bonds are indicated by dotted black lines. TheN-termini of all protein chains are oriented toward the top of thefigure, while the C-termini of all protein chains are oriented towardthe bottom of the Figure. Illustations A-E are bispecific andIllustations F-J are trispecific. Illustations A and E are tetravalent.Illustations B, C, F, I, and J are hexavalent. Illustations D, G, and Hare octavalent. Refer to FIGS. 1, 2, 9, 14 and 17 and to Section 3.1 fordetailed descriptions of the individual domains.

FIG. 36 Binding Ability of HU2B6 4.5-HU3G8 5.1 Biospecific Diabody

FIG. 36 shows the ability of the Hu2B6 4.5-Hu3G8 5.1 biospecific diabody(squares) to bind CD32b and CD16a relative to Hu2B6 4.5 or Hu3G8 5.1diabodies (triangles).

FIG. 37 Schematic of E-Coil and K-Coil DART Derivatives

FIG. 37 illustrates the general conformation of E-coil and K-coil DARTderivatives.

FIG. 38 Helix Arrangement of Preferred E-Coil and K-Coil Separators

FIG. 38 shows the helix arrangement of preferred “E-coil” sequence(EVAALEK)₄ (SEQ ID NO: 299) and preferred “K-coil” sequence (KVAALKE)₄(SEQ ID NO: 300).

FIG. 39 E-Coil and K-Coil Fc-Containing DART Derivatives

FIG. 39 illustrates the different species of E-coil and K-coilFe-containing DART derivatives that can be formed via chain swapping.

FIG. 40 Size Exclusion Chromatography on E-Coil and/or K-CoilDerivatives and E-Coil and/or K-Coil Fc-Containing Derivatives ofh2B6YAhCB3 DARTS

FIG. 40 shows the results of size exclusion chromatography on E-coiland/or K-coil derivatives and E-coil and/or K-coil Fc-containingderivatives of h2B6YAhCB3 DARTS. Four species of such molecules wereanalyzed; all had an E-coli and a K-coil: EK (no Fc region), 2.1 mg;EFc/K (Fc linked to E-coil), 2.7 mgs; E/KFc (Fc linked to K-coil), 1.8mgs; EFc/KFc (Fc linked to the K-coil and the E-coil of the same DART),2.0 mg

FIG. 41 Structure of Produced Dimer Molecules

FIG. 41 shows the possible structure of the produced dimer moleculeidentified in the size exclusion chromatograph of FIG. 40.

FIG. 42 SDS-Polyacrylamide Gel Electrophoretic Analysis of the E-Coiland/or K-Coil Derivatives and E-Coil and/or K-Coil Fc-ContainingDerivatives of h2B6YAhCB3 DARTs

FIG. 42 shows the results of an SDS polyacrylamide gel electrophoreticanalysis of the fractions obtained from size exclusion chromatography(FIG. 40) of E-coil and/or K-coil derivatives and E-coil and/or K-coilFc-containing derivatives of h2B6YAhCB3 DARTs. Flanking lanes: molecularmarker controls; Lane 1: EK (no Fc region); Lane 2: EFc/K, aggregatefraction; Lane 3: EFc/K, monomer fraction; Lane 4: E/KFc, aggregatefraction; Lane 5: E/KFc, monomer fraction; Lane 6: EFc/KFc, aggregatefraction; Lane 7: EFc/KFc, dimer fraction; Lane 8: EFc/KFc, monomerfraction.

FIG. 43 Bispecific Binding ELISA Analysis of

FIG. 43 shows the result of a bispecific binding ELISA comparingE-coil/K-coil Fc-containing h2B6YAhCB3 DART derivatives (EFc/K orE/KFc), h2B6YAhCB3 DART, control and an EFc/KFc h2B6YAhCB3 DARTderivative.

FIG. 44 Ability of the E-Coil and/or K-Coil Derivatives and E-Coiland/or K-coil Fc-Containing Derivatives of h2B6YAhCB3 DARTs to InhibitT-Cell Proliferation

FIG. 44 shows the ability of the E-coil and/or K-coil derivatives andE-coil and/or K-coil Fc-containing derivatives of h2B6YAhCB3 DARTs toinhibit T-cell proliferation.

FIG. 45 hCD16-hCD32B ABD-DART

FIG. 45 shows a schematic of a recombinant antibody molecule,hCD16-hCD32B ABD-DART composed of the ABD3 domain of streptococcalprotein G fused to a recombinant bispecific DART that is immunoreactivewith hCD16 and hCD32B antigens.

FIG. 46A/46B Binding Affinity of hCD16-hCD32B ABD-DART Using DualSpecific ELISA

ELISA plates were coated with either CD16 antigen (FIG. 46A) or humanserum albumin (FIG. 46B) at a concentration of 2 μg/mL. Varyingconcentrations of hCD16-hCD32B ABD-DART (▪) and control hCD16-hCD32BDART (∘) starting with 2 μg/mL were bound. Biotinylated sCD32B antigenwas added to the plate followed by HRP conjugated Streptavidin fordetection.

FIG. 47 PBMC Mediated Cytotoxicity of DART Proteins

PBMC mediated cytotoxicity of DART proteins. ADCC assays were performedusing human B-cell lines, Daudi as target cells incubated with PBMC aseffector cells. Individual assays were done in triplicate at aneffector-to-target ratio of 20:1 and a titration of antibodies:hCD16A-hCD32B DART () and hCD16A-hCD32B ABD DART (▪). Cell mediatedcytotoxicity was measured by LDH release assay. The lower curve at 10°is hCD16A-hCD32B ABD DART (▪).

FIG. 48 Improved Pharmacokinetic Properties of hCD16-hCD32B ABD-DART inC57BI/6 Mice

Mice (n=3) were injected with a single intravenous injection of (A)hCD16-hCD32B ABD-DART () and (B) hCD16-hCD32B DART (▴) at 5 mg/kg.Mouse serum was collected at various time points and concentrations ofprotein in serum were quantified by ELISA. Pharmacokinetic calculationswere performed using WinNonlin Professional 5.1.

FIG. 49A-E HER2×TCR^(b)DART Activity on Panel of HER2 Low ExpressingCell Lines

DART molecules having Her2 and T-cell receptor (TCR) binding domainswere tested for their ability to mediate cytotoxicity in multiple breastcancer, colon cancer and bladder cancer cell lines that had beenpreviously characterized as exhibiting low levels of HER2 expression(and thus being refractory to treatment with the anti-Her2/neu antibody,Herceptin®. The tested breast cancer cell lines are ZR75-1 (HER2 2+)(FIG. 49A), MCF-7 (HER2 1+) (FIG. 49B) and MDA-MB468 (HER2-ve) (FIG.49C). The non-breast cancer cell lines tested are HT-29 (colon cancercell line) (FIG. 49D) and SW780 (bladder cancer cell line) (FIG. 49E).

5. DESCRIPTION OF THE PREFERRED EMBODIMENTS

Each polypeptide chain of the diabody molecule comprises a VL domain anda VH domain, which are covalently linked such that the domains areconstrained from self assembly. Interaction of two of the polypeptidechains will produce two VL-VH pairings, forming two epitope bindingsites, i.e., a bivalent molecule. Neither the VH or VL domain isconstrained to any position within the polypeptide chain, i.e.,restricted to the amino (N) or carboxy (C) terminus, nor are the domainsrestricted in their relative positions to one another, i.e., the VLdomain may be N-terminal to the VH domain and vice-versa. The onlyrestriction is that a complimentary polypeptide chain be available inorder to form functional diabody. Where the VL and VH domains arederived from the same antibody, the two complimentary polypeptide chainsmay be identical. For example, where the binding domains are derivedfrom an antibody specific for epitope A (i.e., the binding domain isformed from a VL_(A)-VH_(A) interaction), each polypeptide will comprisea VH_(A) and a VL_(A). Homodimerization of two polypeptide chains of theantibody will result in the formation two VL_(A)-VH_(A) binding sites,resulting in a bivalent monospecific antibody. Where the VL and VHdomains are derived from antibodies specific for different antigens,formation of a functional bispecific diabody requires the interaction oftwo different polypeptide chains, i.e., formation of a heterodimer. Forexample, for a bispecific diabody, one polypeptide chain will comprise aVL_(A) and a VL_(B); homodimerization of said chain will result in theformation of two VL_(A)-VH_(B) binding sites, either of no binding or ofunpredictable binding. In contrast, where two differing polypeptidechains are free to interact, e.g., in a recombinant expression system,one comprising a VL_(A) and a VH_(B) and the other comprising a VL_(B)and a VH_(A), two differing binding sites will form: VL_(A)-VH_(A) andVL_(B)-VH_(B). For all diabody polypeptide chain pairs, the possibly ofmisalignment or mis-binding of the two chains is a possibility, i.e.,interaction of VL-VL or VH-VH domains; however, purification offunctional diabodies is easily managed based on the immunospecificity ofthe properly dimerized binding site using any affinity based methodknown in the are or exemplified herein, e.g., affinity chromatography.

In other embodiments, one or more of the polypeptide chains of thediabody comprises an Fc domain. Fc domains in the polypeptide chains ofthe diabody molecules preferentially dimerize, resulting in theformation of a diabody molecule that exhibits immunoglobulin-likeproperties, e.g., Fc-FcγR, interactions. Fc comprising diabodies may bedimers, e.g., comprised of two polypeptide chains, each comprising a VHdomain, a VL domain and an Fc domain. Dimerization of said polypeptidechains results in a bivalent diabody comprising an Fc domain, albeitwith a structure distinct from that of an unmodified bivalent antibody(FIG. 11). Such diabody molecules will exhibit altered phenotypesrelative to a wild-type immunoglobulin, e.g., altered serum half-life,binding properties, etc. In other embodiments, diabody moleculescomprising Fc domains may be tetramers. Such tetramers comprise two‘heavier’ polypeptide chains, i.e. a polypeptide chain comprising a VL,aVH and an Fc domain, and two ‘lighter’ polypeptide chains, i.e.,polypeptide chain comprising a VL and a VH. Said lighter and heavierchains interact to form a monomer, and said monomers interact via theirunpaired Fc domains to form an Ig-like molecule. Such an Ig-like diabodyis tetravalent and may be monospecific, bispecific or tetraspecific.

The at least two binding sites of the diabody molecule can recognize thesame or different epitopes. Different epitopes can be from the sameantigen or epitopes from different antigens. In one embodiment, theepitopes are from different cells. In another embodiment, the epitopesare cell surface antigens on the same cell or virus. The epitopesbinding sites can recognize any antigen to which an antibody can begenerated. For example, proteins, nucleic acids, bacterial toxins, cellsurface markers, autoimmune markers, viral proteins, drugs, etc. Inparticular aspects, at least one epitope binding site of the diabody isspecific for an antigen on a particular cell, such as a B-cell orT-cell, a phagocytotic cell, a natural killer (NK) cell or a dendriticcell.

Each domain of the polypeptide chain of the diabody, i.e., the VL, VHand FC domain may be separated by a peptide linker. The peptide linkermay be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. In certainembodiments the amino acid linker sequence is GGGSGGGG (SEQ ID NO: 10)encoded by the nucleic acid sequence (SEQ ID NO: 74).

In certain embodiments, each polypeptide chain of the diabody moleculeis engineered to comprise at least one cysteine residue that willinteract with a counterpart at least one cysteine residue on a secondpolypeptide chain of the invention to form an inter-chain disulfidebond. Said interchain disulfide bonds serve to stabilize the diabodymolecule, improving expression and recovery in recombinant systems,resulting in a stable and consistent formulation as well as improvingthe stability of the isolated and/or purified product in vivo. Said atleast one cysteine residue may be introduced as a single amino acid oras part of larger amino-acid sequence, e.g. hinge domain, in any portionof the polypeptide chain. In a specific embodiment, said at least onecysteine residue is engineered to occur at the C-terminus of thepolypeptide chain. In some embodiments, said at least one cysteineresidue in introduced into the polypeptide chain within the amino acidsequence LGGC. In a specific embodiment, the C-terminus of thepolypeptide chain comprising the diabody molecule of the inventioncomprises the amino acid sequence LGGC. In another embodiment, said atleast one cysteine residue is introduced into the polypeptide within anamino acid sequence comprising a hinge domain, e.g. SEQ ID NO: 1 or SEQID NO: 4. In a specific embodiment, the C-terminus of a polypeptidechain of the diabody molecule of the invention comprises the amino acidsequence of an IgG hinge domain, e.g. SEQ ID NO: 1. In anotherembodiment, the C-terminus of a polypeptide chain of a diabody moleculeof the invention comprises the amino acid sequence VEPKSC (SEQ ID NO:77), which can be encoded by nucleotide sequence (SEQ ID NO: 78). Inother embodiments, said at least one cysteine residue in introduced intothe polypeptide chain within the amino acid sequence LGGCFNRGEC (SEQ IDNO: 17), which can be encoded by the nucleotide sequence (SEQ ID NO:76). In a specific embodiment, the C-terminus of a polypeptide chaincomprising the diabody of the invention comprises the amino acidsequence LGGCFNRGEC (SEQ ID NO: 17), which can be encoded by thenucleotide sequence (SEQ ID NO: 76). In yet other embodiments, said atleast one cysteine residue in introduced into the polypeptide chainwithin the amino acid sequence FNRGEC (SEQ ID NO: 23), which can beencoded by the nucleotide sequence (SEQ ID NO: 75). In a specificembodiment, the C-terminus of a polypeptide chain comprising the diabodyof the invention comprises the amino acid sequence FNRGEC (SEQ ID NO:23), which can be encoded by the nucleotide sequence (SEQ ID NO: 75).

In certain embodiments, the diabody molecule comprises at least twopolypeptide chains, each of which comprise the amino acid sequence LGGCand are covalently linked by a disulfide bond between the cysteineresidues in said LGGC sequences. In another specific embodiment, thediabody molecule comprises at least two polypeptide chains, one of whichcomprises the sequence FNRGEC (SEQ ID NO: 23) while the other comprisesa hinge domain (containing at least one cysteine residue), wherein saidat least two polypeptide chains are covalently linked by a disulfidebond between the cysteine residue in FNRGEC (SEQ ID NO: 23) and acysteine residue in the hinge domain. In particular aspects, thecysteine residue responsible for the disulfide bond located in the hingedomain is Cys-128 (as numbered according to Kabat EU; located in thehinge domain of an unmodified, intact IgG heavy chain) and thecounterpart cysteine residue in SEQ ID NO: 23 is Cys-214 (as numberedaccording to Kabat EU; located at the C-terminus of an unmodified,intact IgG light chain) (Elkabetz et al. (2005) “Cysteines In CH1Underlie Retention Of Unassembled Ig Heavy Chains,” J. Biol. Chem.280:14402-14412; hereby incorporated by reference herein in itsentirety). In yet other embodiments, the at least one cysteine residueis engineered to occur at the N-terminus of the amino acid chain. Instill other embodiments, the at least one cysteine residue is engineeredto occur in the linker portion of the polypeptide chain of the diabodymolecule. In further embodiments, the VH or VL domain is engineered tocomprise at least one amino acid modification relative to the parentalVH or VL domain such that said amino acid modification comprises asubstitution of a parental amino acid with cysteine.

The invention encompasses diabody molecules comprising an Fc domain orportion thereof (e.g. a CH2 domain, or CH3 domain). The Fc domain orportion thereof may be derived from any immunoglobulin isotype orallotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. Inpreferred embodiments, the Fc domain (or portion thereof) is derivedfrom IgG. In specific embodiments, the IgG isotype is IgG1, IgG2, IgG3or IgG4 or an allotype thereof. In one embodiment, the diabody moleculecomprises an Fc domain, which Fc-domain comprises a CH2 domain and CH3domain independently selected from any immunoglobulin isotype (i.e. anFc domain comprising the CH2 domain derived from IgG and the CH3 domainderived form IgE, or the CH2 domain derived from IgG 1 and the CH3domain derived from IgG2, etc.). Said Fc domain may be engineered into apolypeptide chain comprising the diabody molecule of the invention inany position relative to other domains or portions of said polypeptidechain (e.g., the Fc domain, or portion thereof, may be c-terminal toboth the VL and VH domains of the polypeptide of the chain; may ben-terminal to both the VL and VH domains; or may be N-terminal to onedomain and c-terminal to another (i.e., between two domains of thepolypeptide chain)).

The present invention also encompasses molecules comprising a hingedomain. The hinge domain be derived from any immunoglobulin isotype orallotype including IgA, IgD, IgG, IgE and IgM. In preferred embodiments,the hinge domain is derived from IgG, wherein the IgG isotype is IgG1,IgG2, IgG3 or IgG4, or an allotpye thereof. Said hinge domain may beengineered into a polypeptide chain comprising the diabody moleculetogether with an Fc domain such that the diabody molecule comprises ahinge-Fc domain. In certain embodiments, the hinge and Fc domain areindependently selected from any immunoglobulin isotype known in the artor exemplified herein. In other embodiments the hinge and Fc domain areseparated by at least one other domain of the polypeptide chain, e.g.,the VL domain. The hinge domain, or optionally the hinge-Fc domain, maybe engineered in to a polypeptide of the invention in any positionrelative to other domains or portions of said polypeptide chain. Incertain embodiments, a polypeptide chain of the invention comprises ahinge domain, which hinge domain is at the C-terminus of the polypeptidechain, wherein said polypeptide chain does not comprise an Fc domain. Inyet other embodiments, a polypeptide chain of the invention comprises ahinge-Fc domain, which hinge-Fc domain is at the C-terminus of thepolypeptide chain. In further embodiments, a polypeptide chain of theinvention comprises a hinge-Fc domain, which hinge-Fc domain is at theN-terminus of the polypeptide chain.

As discussed above, the invention encompasses multimers of polypeptidechains, each of which polypeptide chains comprise a VH and VL domain. Incertain aspects, the polypeptide chains in said multimers furthercomprise an Fc domain. Dimerization of the Fc domains leads to formationof a diabody molecule that exhibits immunoglobulin-like functionality,i.e., Fc mediated function (e.g., Fc-FcγR interaction, complementbinding, etc.). In certain embodiments, the VL and VH domains comprisingeach polypeptide chain have the same specificity, and said diabodymolecule is bivalent and monospecific. In other embodiments, the VL andVH domains comprising each polypeptide chain have differing specificityand the diabody is bivalent and bispecific.

In yet other embodiments, diabody molecules of the invention encompasstetramers of polypeptide chains, each of which polypeptide chaincomprises a VH and VL domain. In certain embodiments, two polypeptidechains of the tetramer further comprise an Fc domain. The tetramer istherefore comprised of two ‘heavier’ polypeptide chains, each comprisinga VL, VH and Fc domain, and two ‘lighter’ polypeptide chains, comprisinga VL and VH domain. Interaction of a heavier and lighter chain into abivalent monomer coupled with dimerization of said monomers via the Fcdomains of the heavier chains will lead to formation of a tetravalentimmunoglobulin-like molecule (exemplified in Example 6.2 and Example6.3). In certain aspects the monomers are the same, and the tetravalentdiabody molecule is monospecific or bispecific. In other aspects themonomers are different, and the tetra valent molecule is bispecific ortetraspecific.

Formation of a tetraspecific diabody molecule as described suprarequires the interaction of four differing polypeptide chains. Suchinteractions are difficult to achieve with efficiency within a singlecell recombinant production system, due to the many variants ofpotential chain mispairings. One solution to increase the probability ofmispairings, is to engineer “knobs-into-holes” type mutations into thedesired polypeptide chain pairs. Such mutations favor heterodimerizationover homodimerization. For example, with respect to Fc-Fc-interactions,an amino acid substitution (preferably a substitution with an amino acidcomprising a bulky side group forming a ‘knob’, e.g., tryptophan) can beintroduced into the CH2 or CH3 domain such that steric interference willprevent interaction with a similarly mutated domain and will obligatethe mutated domain to pair with a domain into which a complementary, oraccommodating mutation has been engineered, i.e., ‘the hole’ (e.g., asubstitution with glycine). Such sets of mutations can be engineeredinto any pair of polypeptides comprising the diabody molecule, andfurther, engineered into any portion of the polypeptides chains of saidpair. Methods of protein engineering to favor heterodimerization overhomodimerization are well known in the art, in particular with respectto the engineering of immunoglobulin-like molecules, and are encompassedherein (see e.g., Ridgway et al. (1996) “Knobs-Into-Holes' EngineeringOf Antibody CH3 Domains For Heavy Chain Heterodimerization,” ProteinEngr. 9:617-621, Atwell et al. (1997) “Stable Heterodimers FromRemodeling The Domain Interface Of A Homodimer Using A Phage DisplayLibrary,” J. Mol. Biol. 270: 26-35, and Xie et al. (2005) “A New FormatOf Bispecific Antibody: Highly Efficient Heterodimerization, ExpressionAnd Tumor Cell Lysis,” J. Immunol. Methods 296:95-101; each of which ishereby incorporated herein by reference in its entirety).

The invention also encompasses diabody molecules comprising variant Fcor variant hinge-Fc domains (or portion thereof), which variant Fcdomain comprises at least one amino acid modification (e.g.substitution, insertion deletion) relative to a comparable wild-type Fcdomain or hinge-Fc domain (or portion thereof). Molecules comprisingvariant Fc domains or hinge-Fc domains (or portion thereof) (e.g.,antibodies) normally have altered phenotypes relative to moleculescomprising wild-type Fc domains or hinge-Fc domains or portions thereof.The variant phenotype may be expressed as altered serum half-life,altered stability, altered susceptibility to cellular enzymes or alteredeffector function as assayed in an NK dependent or macrophage dependentassay. Fc domain variants identified as altering effector function aredisclosed in International Application WO04/063351, U.S. PatentApplication Publications 2005/0037000 and 2005/0064514, U.S. ProvisionalApplications 60/626,510, filed Nov. 10, 2004, 60/636,663, filed Dec. 15,2004, and 60/781,564, filed Mar. 10, 2006, and U.S. patent applicationSer. Nos. 11/271,140, filed Nov. 10, 2005, and 11/305,787, filed Dec.15, 2005, concurrent applications of the Inventors, each of which isincorporated by reference in its entirety.

The bispecific diabodies of the invention can simultaneously bind twoseparate and distinct epitopes. In certain embodiments the epitopes arefrom the same antigen. In other embodiments, the epitopes are fromdifferent antigens. In preferred embodiments, at least one epitopebinding site is specific for a determinant expressed on an immuneeffector cell (e.g. CD3, CD16, CD32, CD64, etc.) which are expressed onT lymphocytes, natural killer (NK) cells or other mononuclear cells. Inone embodiment, the diabody molecule binds to the effector celldeterminant and also activates said effector cell. In this regard,diabody molecules of the invention may exhibit Ig-like functionalityindependent of whether they further comprise an Fc domain (e.g., asassayed in any effector function assay known in the art or exemplifiedherein (e.g., ADCC assay). In certain embodiments the bispecific diabodyof the invention binds both a cancer antigen on a tumor cell and aneffector cell determinant while activating said cell. In alternativeembodiments, the bispecific diabody or diabody molecule of the inventionmay inhibit activation of a target, e.g., effector, cell bysimultaneously binding, and thus linking, an activating and inhibitoryreceptor on the same cell (e.g., bind both CD32A and CD32B, BCR andCD32B, or IgER1 and CD32B) as described supra (see, Background Section).In a further aspect of this embodiment, the bispecific diabody mayexhibit anti-viral properties by simultaneously binding two neutralizingepitopes on a virus (e.g., RSV epitopes; WNV epitopes such as E16 andE53).

In certain embodiments, bispecific diabody molecules of the inventionoffer unique opportunities to target specific cell types. For example,the bispecific diabody or diabody molecule can be engineered to comprisea combination of epitope binding sites that recognize a set of antigensunique to a target cell or tissue type. Additionally, where either orboth of the individual antigens is/are fairly common separately in othertissue and/or cell types, low affinity biding domains can be used toconstruct the diabody or diabody molecule. Such low affinity bindingdomains will be unable to bind to the individual epitope or antigen withsufficient avidity for therapeutic purposes. However, where bothepitopes or antigens are present on a single target cell or tissue, theavidity of the diabody or diabody molecule for the cell or tissue,relative to a cell or tissue expressing only one of the antigens, willbe increased such that said cell or tissue can be effectively targetedby the invention. Such a bispecific molecule can exhibit enhancedbinding to one or both of its target antigens on cells expressing bothof said antigens relative to a monospecific diabody or an antibody witha specificity to only one of the antigens.

Preferably, the binding properties of the diabodies of the invention arecharacterized by in vitro functional assays for determining bindingactivity and/or one or more FcγR mediator effector cell functions(mediated via Fc-FcγR interactions or by the immunospecific binding of adiabody molecule to an FcγR) (See Section 5.4.2 and 5.4.3). Theaffinities and binding properties of the molecules, e.g., diabodies, ofthe invention for an FcγR can be determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining binding domain-antigen or Fc-FcγR interactions, i.e.,specific binding of an antigen to a binding domain or specific bindingof an Fc region to an FcγR, respectively, including but not limited toELISA assay, surface plasmon resonance assay, immunoprecipitation assays(See Section 5.4.2). In most preferred embodiments, the molecules of theinvention have similar binding properties in in vivo models (such asthose described and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

In some embodiments, molecules of the invention are engineered tocomprise an altered glycosylation pattern or an altered glycoformrelative to the comparable portion of the template molecule. Engineeredglycoforms may be useful for a variety of purposes, including, but notlimited to, enhancing effector function. Engineered glycoforms may begenerated by any method known to one skilled in the art, for example byusing engineered or variant expression strains, by co-expression withone or more enzymes, for example, DI N-acetylglucosaminyltransferase III(GnTIII), by expressing a diabody of the invention in various organismsor cell lines from various organisms, or by modifying carbohydrate(s)after the diabody has been expressed and purified. Methods forgenerating engineered glycoforms are known in the art, and include butare not limited to those described in Umana et al. (1999) “EngineeredGlycoforms Of An Antineuroblastoma IgG1 With OptimizedAntibody-Dependent Cellular Cytotoxic Activity,” Nat. Biotechnol17:176-180; Davies et al. (2001) “Expression Of GnTIII In A RecombinantAnti-CD20 CHO Production Cell Line: Expression Of Antibodies WithAltered Glycoforms Leads To An Increase In Adcc Through Higher AffinityFor Fc Gamma RIII,” Biotechnol Bioeng 74:288-294; Shields et al. (2002)“Lack Of Fucose On Human IgG1 N-Linked Oligosaccharide Improves BindingTo Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,” J BiolChem 277:26733-26740; Shinkawa et al. (2003) “The Absence Of Fucose ButNot The Presence Of Galactose Or Bisecting N-Acetylglucosamine Of HumanIgG1 Complex-Type Oligosaccharides Shows The Critical Role Of EnhancingAntibody-Dependent Cellular Cytotoxicity,” J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1;PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton,N.J.); GlycoMAb™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland); each of which is incorporatedherein by reference in its entirety. See, e.g., WO 00061739; EA01229125;US 20030115614; Okazaki et al. (2004) “Fucose Depletion From Human IgG1Oligosaccharide Enhances Binding Enthalpy And Association Rate BetweenIgG1 And FcGammaRIIIA,” JMB, 336: 1239-49 each of which is incorporatedherein by reference in its entirety.

The invention further encompasses incorporation of unnatural amino acidsto generate the diabodies of the invention. Such methods are known tothose skilled in the art such as those using the natural biosyntheticmachinery to allow incorporation of unnatural amino acids into proteins,see, e.g., Wang et al. (2002) “Expanding The Genetic Code,” Chem. Comm.1:1-11; Wang et al. (2001) “Expanding The Genetic Code Of Escherichiacoli,” Science, 292: 498-500; van Hest et al. (2001) “Protein-BasedMaterials, Toward A New Level Of Structural Control,” Chem. Comm. 19:1897-1904, each of which is incorporated herein by reference in itsentirety. Alternative strategies focus on the enzymes responsible forthe biosynthesis of amino acyl-tRNA, see, e.g., Tang et al. (2001)“Biosynthesis Of A Highly Stable Coiled-Coil Protein ContainingHexafluoroleucine In An Engineered Bacterial Host,” J. Am. Chem. Soc.123(44): 11089-11090; Kiick et al. (2001) “Identification Of An ExpandedSet Of Translationally Active Methionine Analogues In Escherichia coli,”FEBS Lett. 502(1-2):25-30; each of which is incorporated herein byreference in its entirety.

In some embodiments, the invention encompasses methods of modifying aVL, VH or Fc domain of a molecule of the invention by adding or deletinga glycosylation site. Methods for modifying the carbohydrate of proteinsare well known in the art and encompassed within the invention, see,e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S. Publication No. US2002/0028486; WO 03/035835; U.S. Publication No. 2003/0115614; U.S. Pat.No. 6,218,149; U.S. Pat. No. 6,472,511; all of which are incorporatedherein by reference in their entirety.

The diabody molecules of the present invention may be constructed tocomprise a domain that is a binding ligand for the Natural Killer Group2D (NKG2D) receptor. Such binding ligands, and particularly those whichare not expressed on normal cells, include the histocompatibility 60(H60) molecule, the product of the retinoic acid early inducible gene-1(RAE-1), and the murine UL16-binding proteinlike transcript 1 (MULTI)(Raulet D. H. (2003) “Roles Of The NKG2D Immunoreceptor And ItsLigands,” Nature Rev. Immunol. 3:781-790; Coudert, J. D. et al. (2005)“Altered NKG2D Function In NK Cells Induced By Chronic Exposure ToAltered NKG2D Ligand-Expressing Tumor Cells,” Blood 106:1711-1717).Additional ligands reactive with human NKG2D include the polymorphic MHCclass I chain-related molecules MICA and MICB (Diefenbach, A. et al.(1999) “Natural Killer Cells: Stress Out, Turn On, Tune In,” Curr. Biol.9(22):R851-R8533; Bauer, S. et al. (1999) “Activation Of NK Cells And TCells By NKG2D, A Receptor For Stress-Inducible MICA,” Science285(5428):727-729; Stephens, H. A. (2001) “MICA and MICB genes: can theenigma of their polymorphism be resolved?,” Trends Immunol. 22:378-385.

The sequence of MICA is SEQ ID NO: 311:

MGLGPVFLLL AGIFPFAPPG AAAEPHSLRY NLTVLSWDGS VQSGFLTEVH LDGQPFLRCDRQKCRAKPQG QWAEDVLGNK TWDRETRDLT GNGKDLRMTL AHIKDQKEGL HSLQEIRVCEIHEDNSTRSS QHFYYDGELF LSQNLETKEW TMPQSSRAQT LAMNVRNFLK EDAMKTKTHYHAMHADCLQE LRRYLKSGVV LRRTVPPMVN VTRSEASEGN ITVTCRASGF YPWNITLSWRQDGVSLSHDT QQWGDVLPDG NGTYQTWVAT RICQGEEQRF TCYMEHSGNH STHPVPSGKVLVLQSHWQTF HVSAVAAAAI FVIIIFYVRC CKKKTSAAEG PELVSLQVLD QHPVGTSDHRDATQLGFQPL MSDLGSTGST EGA

The sequence of MICB is SEQ ID NO: 312:

PHSLRYNLMV LSQDGSVQSG FLAEGHLDGQ PFLRYDRQKR RAKPQGQWAE DVLGAKTWDTETEDLTENGQ DLRRTLTHIK DQKGGLHSLQ EIRVCEIHED SSTRGSRHFY YDGELFLSQNLETQESTVPQ SSRAQTLAMN VTNFWKEDAM KTKTHYRAMQ ADCLQKLQLP PMVNVICSEVSEGNITVTCR ASSFYPRNIT LTWRQDGVSL SHNTQQWGDV LPDGNGTYQT WVATRIRQGEEQRFTCYMEH SGNHGTHPVP SGKALVLQSQ RTDFPYVSAA MPCFVIIIIL CVPCCKKKTS AAEGP

Antibodies that specifically bind to the T-cell Receptor include theanti-TCR antibody BMA 031 (Kurrle, R. et al. (1989) “BMA 031—ATCR-Specific Monoclonal Antibody For Clinical Application,” TransplantProc. 21(1 Pt 1):1017-1019; Nashan, B. et al. (1987) “Fine SpecificityOf A Panel Of Antibodies Against The TCR/CD3 Complex,” Transplant Proc.19(5):4270-4272; Shearman, C. W. et al. (1991) “Construction,Expression, And Biologic Activity Of Murine/Human Chimeric AntibodiesWith Specificity For The Human α/β T Cell,” J. Immunol. 146(3):928-935;Shearman, C. W. et al. (1991) “Construction, Expression AndCharacterization of Humanized Antibodies Directed Against The Human α/βT Cell Receptor,” J. Immunol. 147(12):4366-4373). Antibodies thatspecifically bind to the NKG2D Receptor include KYK-2.0 (Kwong, K Y etal. (2008) “Generation, Affinity Maturation, And Characterization Of AHuman Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic AndAgonistic Activity,” J. Mol. Biol. 384:1143-1156; and PCT/US09/54911).

Through the use of such a diabody, the target cell is now redirected tobe a cell that can be bound by cells that array the (NKG2D) receptor.The NKG2D receptor is expressed on all human (and other mammalian)Natural Killer cells (Bauer, S. et al. (1999) “Activation Of NK CellsAnd T Cells By NKG2D, A Receptor For Stress-Inducible MICA,” Science285(5428):727-729; Jamieson, A. M. et al. (2002) “The Role Of The NKG2DImmunoreceptor In Immune Cell Activation And Natural Killing,” Immunity17(1):19-29) as well as on all CD8⁺ T cells (Groh, V. et al. (2001)“Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC InducedOn Virus-Infected Cells,” Nat. Immunol. 2(3):255-260; Jamieson, A. M. etal. (2002) “The Role Of The NKG2D Immunoreceptor In Immune CellActivation And Natural Killing,” Immunity 17(1):19-29).

Alternatively, the diabody molecules of the present invention may beconstructed to comprise a domain that is a binding ligand for the T-cellreceptor (“TCR”). The TCR is natively expressed by CD4+ or CD8+ T-cells,and permits such cells to recognize antigenic peptides that are boundand presented by class I or class II MHC proteins of antigen-presentingcells. Recognition of a pMHC (peptide—MHC) complex by a TCR initiatesthe propagation of a cellular immune response that leads to theproduction of cytokines and the lysis of the antigen-presenting cell(see, e.g., Armstrong, K. M. et al. (2008) “Conformational Changes AndFlexibility In T-Cell Receptor Recognition Of Peptide—MHC Complexes,”Biochem. J. 415(Pt 2):183-196; Willemsen, R. (2008) “Selection Of HumanAntibody Fragments Directed Against Tumor T-Cell Epitopes For AdoptiveT-Cell Therapy,” Cytometry A. 73(11):1093-1099; Beier, K. C. et al.(2007) “Master Switches Of T-Cell Activation And Differentiation,” Eur.Respir. J. 29:804-812; Mallone, R. et al. (2005) “Targeting TLymphocytes For Immune Monitoring And Intervention In AutoimmuneDiabetes,” Am. J. Ther. 12(6):534-550).

By constructing such diabody molecules to additionally comprise at leastone epitope-binding domain capable of binding to, for example, areceptor present on the surface of a target cell, such diabody moleculeswill be DART molecules and thus be capable of binding to the targetcells and thereby cause the target cells to display the binding ligandfor the Natural Killer Group 2D (NKG2D) receptor or to the TCR(whichever is present on the target cell-bound diabody) (see, e.g.,Germain, C. et al. (2008) “Redirecting NK Cells Mediated Tumor CellLysis By A New Recombinant Bifunctional Protein,” Prot. Engineer.Design. Selection 21(11):665-672).

Such diabodies can be used to redirect any desired target cell into acell that is a target of NK cell-mediated cell lysis or T-cell mediatedcytotoxicity. In one embodiment, the epitope-binding domain of thediabody capable of binding to a receptor present on the surface of atarget cell is an epitope that binds to a tumor-associated antigen so asto redirect such cancer cells into substrates for NK cell-mediated celllysis or T-cell mediated cytotoxicity. Of particular interest is atumor-associated antigens that is a breast cancer antigen, an ovariancancer antigen, a prostate cancer antigen, a cervical cancer antigen, apancreatic carcinoma antigen, a lung cancer antigen, a bladder cancerantigen, a colon cancer antigen, a testicular cancer antigen, aglioblastoma cancer antigen, an antigen associated with a B cellmalignancy, an antigen associated with multiple myeloma, an antigenassociated with non-Hodgkins lymphoma, or an antigen associated withchronic lymphocytic leukemia.

Suitable tumor-associated antigens for such use include A33 (acolorectal carcinoma antigen; Almqvist, Y. 2006, Nucl Med. Biol.November; 33(8):991-998); B1 (Egloff, A. M. et al. 2006, Cancer Res.66(1):6-9); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84);beta-catenin (Prange W. et al. 2003 J Pathol. 201(2):250-9); CA125(Bast, R. C. Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81);CD5 (Calin, G. A. et al. 2006 Semin Oncol. 33(2):167-73; CD19(Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD20(Thomas, D. A. et al. 2006 Hematol Oncol Clin North Am. 20(5):1125-36);CD22 (Kreitman, R. J. 2006 AAPS J. 18; 8(3):E532-51); CD23 (Rosati, S.et al. 2005 Curr Top Microbiol Immunol. 5; 294:91-107); CD25 (Troussard,X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD27 (Bataille, R. 2006Haematologica 91(9):1234-40); CD28 (Bataille, R. 2006 Haematologica91(9):1234-40); CD36 (Ge, Y. 2005 Lab Hematol. 11(1):31-7); CD40/CD154(Messmer, D. et al. 2005 Ann N Y Acad. Sci. 1062:51-60); CD45 (Jurcic,J. G. 2005 Curr Oncol Rep. 7(5):339-46); CD56 (Bataille, R. 2006Haematologica 91(9):1234-40); CD79a/CD79b (Troussard, X. et al. 1998Hematol Cell Ther. 40(4):139-48; Chu, P. G. et al. 2001 ApplImmunohistochem Mol Morphol. 9(2):97-106); CD103 (Troussard, X. et al.1998 Hematol Cell Ther. 40(4):139-48); CDK4 (Lee, Y. M. et al. 2006 CellCycle 5(18):2110-4); CEA (carcinoembryonic antigen; Mathelin, C. 2006Gynecol Obstet. Fertil. 34(7-8):638-46; Tellez-Avila, F. I. et al. 2005Rev Invest Clin. 57(6):814-9); CTLA4 (Peggs, K. S. et al. 2006 Curr OpinImmunol. 18(2):206-13); EGF-R (epidermal growth factor receptor; Adenis,A. et al. 2003 Bull Cancer. 90 Spec No:S228-32); Erb (ErbB1; ErbB3;ErbB4; Zhou, H. et al. 2002 Oncogene 21(57):8732-40; Rimon, E. et al.2004 Int J. Oncol. 24(5):1325-38); GAGE (GAGE-1; GAGE-2; Akcakanat, A.et al. 2006 Int J. Cancer. 118(1):123-8); GD2/GD3/GM2 (Livingston, P. O.et al. 2005 Cancer Immunol Immunother. 54(10):1018-25); gp100 (Lotem, M.et al. 2006 J Immunother. 29(6):616-27); HER-2/neu (Kumar, Pal S et al.2006 Semin Oncol. 33(4):386-91); human papillomavirus-E6/humanpapillomavirus-E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; KSA(17-1A) (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); MAGE (MAGE-1;MAGE-3; (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); MART(Kounalakis, N. et al. 2005 Curr Oncol Rep. 7(5):377-82; MUC-1(Mathelin, C. 2006 Gynecol Obstet. Fertil. 34(7-8):638-46); MUM-1(Castelli, C. et al. 2000 J Cell Physiol. 182(3):323-31);N-acetylglucosaminyltransferase (Dennis, J. W. 1999 Biochim BiophysActa. 6; 1473(1):21-34); p 15 (Gil, J. et al. 2006 Nat Rev Mol CellBiol. 7(9):667-77); PSA (prostate specific antigen; Cracco, C. M. et al.2005 Minerva Urol Nefrol. 57(4):301-11); PSMA (Ragupathi, G. 2005 CancerTreat Res. 123:157-80); sTn (Holmberg, L. A. 2001 Expert Opin Biol Ther.1(5):881-91); TNF-receptor (TNF-α receptor, TNF-β receptor; or TNF-γreceptor; van Horssen, R. et al. 2006 Oncologist. 11(4):397-408;Gardnerova, M. et al. 2000 Curr Drug Targets. 1(4):327-64); or VEGFreceptor (O'Dwyer. P. J. 2006 Oncologist. 11(9):992-8).

Additional tumor-associated antigens for such use (and publicationsdisclosing specifically reactive antibodies for such antigens) includeADAM-9 (United States Patent Publication No. 2006/0172350; PCTPublication No. WO 06/084075); ALCAM (PCT Publication No. WO 03/093443);Carboxypeptidase M (United States Patent Publication No. 2006/0166291);CD46 (U.S. Pat. No. 7,148,038; PCT Publication No. WO 03/032814);Cytokeratin 8 (PCT Publication No. WO 03/024191); Ephrin receptors (andin particular EphA2 (U.S. Pat. No. 7,569,672; PCT Publication No. WO06/084226); Integrin Alpha-V-Beta-6 (PCT Publication No. WO 03/087340);JAM-3 (PCT Publication No. WO 06/084078); KID3 (PCT Publication No. WO05/028498); KID31 (PCT Publication No. WO 06/076584); LUCA-2 (UnitedStates Patent Publication No. 2006/0172349; PCT Publication No. WO06/083852); Oncostatin M (Oncostatin Receptor Beta) (U.S. Pat. No.7,572,896; PCT Publication No. WO 06/084092); PIPA (U.S. Pat. No.7,405,061; PCT Publication No. WO 04/043239); RAAG10 (U.S. Pat. No.7,527,969; PCT Publication No. WO 04/001381); ROR1 (U.S. Pat. No.5,843,749); TEST (PCT Publication No. WO 08/066,691); and theTransferrin Receptor (U.S. Pat. No. 7,572,895; PCT Publication No. WO05/121179).

Also of interest are antigens specific to particular infectious agents,e.g., viral agents including, but not limited to human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), influenza, human papilloma virus(HPV), foot and mouth (coxsackieviruses), the rabies virus, herpessimplex virus (HSV), and the causative agents of gastroenteritis,including rotaviruses, adenoviruses, caliciviruses, astroviruses andNorwalk virus; bacterial agents including, but not limited to E. coli,Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae,Neisseria gonorrhoeae, Helicobacter pylori, Hemophilus influenzae,Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosisand Streptococcus pneumoniae, fungal agents and parasites such asGiardi.

Alternatively, such epitope may bind to an Fc receptor (e.g., FcγRI orFcγRII), so as to, for example redirect acute monocytic leukemic cellsinto substrates for NK cell-mediated cell lysis.

5.1 Diabody Binding Domains

The diabodies of the present invention comprise antigen binding domainsgenerally derived from immunoglobulins or antibodies. The antibodiesfrom which the binding domains used in the methods of the invention arederived may be from any animal origin including birds and mammals (e.g.,human, non-human primate, murine, donkey, sheep, rabbit, goat, guineapig, camel, horse, or chicken). Preferably, the antibodies are human orhumanized monoclonal antibodies. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or libraries of synthetic human immunoglobulin codingsequences or from mice that express antibodies from human genes.

The invention contemplates the use of any antibodies known in the artfor the treatment and/or prevention of cancer, autoimmune disease,inflammatory disease or infectious disease as source of binding domainsfor the diabodies of the invention. Non-limiting examples of knowncancer antibodies are provided in section 5.7.1 as well as otherantibodies specific for the listed target antigens and antibodiesagainst the cancer antigens listed in section 5.6.1; nonlimitingexamples of known antibodies for the treatment and/or prevention ofautoimmune disease and inflammatory disease are provided in section5.7.2, as well as antibodies against the listed target antigens andantibodies against the antigens listed in section 5.6.2; in otherembodiments antibodies against epitopes associated with infectiousdiseases as listed in Section 5.6.3 can be used. In certain embodiments,the antibodies comprise a variant Fc region comprising one or more aminoacid modifications, which have been identified by the methods of theinvention to have a conferred effector function and/or enhanced affinityfor FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild type Fc region. A non-limitingexample of the antibodies that are used for the treatment or preventionof inflammatory disorders which can be engineered according to theinvention is presented in Table 9, and a non-limiting example of theantibodies that are used for the treatment or prevention of autoimmunedisorder is presented in Table 10.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use diabodies withvariable domains derived from human, chimeric or humanized antibodies.Variable domains from completely human antibodies are particularlydesirable for therapeutic treatment of human subjects. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

A humanized antibody is an antibody, a variant or a fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodymay comprise substantially all of at least one, and typically two,variable domains in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the donorantibody. Such mutations, however, are preferably not extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental framework region (FR) and CDR sequences, moreoften 90%, and most preferably greater than 95%. Humanized antibodiescan be produced using variety of techniques known in the art, includingbut not limited to, CDR-grafting (European Patent No. EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (European PatentNos. EP 592,106 and EP 519,596; Padlan (1991) “A Possible Procedure ForReducing The Immunogenicity Of Antibody Variable Domains WhilePreserving Their Ligand-Binding Properties,” Molecular Immunology28(4/5):489-498; Studnicka et al. (1994) “Human-Engineered MonoclonalAntibodies Retain Full Specific Binding Activity By Preserving Non-CDRComplementarity-Modulating Residues,” Protein Engineering 7(6):805-814;and Roguska et al. (1994) “Humanization Of Murine Monoclonal AntibodiesThrough Variable Domain Resurfacing,” Proc Natl Acad Sci USA91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniquesdisclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089,International Publication No. WO 9317105, Tan et al. (2002)“‘Superhumanized’ Antibodies: Reduction Of Immunogenic Potential ByComplementarity-Determining Region Grafting With Human GermlineSequences: Application To An Anti-CD28,” J. Immunol. 169:1119-25, Caldaset al. (2000) “Design And Synthesis Of Germline-Based Hemi-HumanizedSingle-Chain Fv Against The CD18 Surface Antigen,” Protein Eng.13:353-60, Morea et al. (2000) “Antibody Modeling: Implications ForEngineering And Design,” Methods 20:267-79, Baca et al. (1997) “AntibodyHumanization Using Monovalent Phage Display,” J. Biol. Chem.272:10678-84, Roguska et al. (1996) “A Comparison Of Two MurineMonoclonal Antibodies Humanized By CDR-Grafting And Variable DomainResurfacing,” Protein Eng. 9:895-904, Couto et al. (1995) “DesigningHuman Consensus Antibodies With Minimal Positional Templates,” CancerRes. 55 (23 Supp):5973s-5977s, Couto et al. (1995) “Anti-BA46 MonoclonalAntibody Mc3: Humanization Using A Novel Positional Consensus And InVivo And In Vitro Characterization,” Cancer Res. 55:1717-22, Sandhu(1994) “A Rapid Procedure For The Humanization Of MonoclonalAntibodies,” Gene 150:409-10, Pedersen et al. (1994) “Comparison OfSurface Accessible Residues In Human And Murine Immunoglobulin FvDomains. Implication For Humanization Of Murine Antibodies,” J. Mol.Biol. 235:959-973, Jones et al. (1986) “Replacing TheComplementarity-Determining Regions In A Human Antibody With Those FromA Mouse,” Nature 321:522-525, Riechmann et al. (1988) “Reshaping HumanAntibodies For Therapy,” Nature 332:323-327, and Presta (1992) “AntibodyEngineering,” Curr. Op. Biotech. 3(4):394-398. Often, framework residuesin the framework regions will be substituted with the correspondingresidue from the CDR donor antibody to alter, preferably improve,antigen binding. These framework substitutions are identified by methodswell known in the art, e.g., by modeling of the interactions of the CDRand framework residues to identify framework residues important forantigen binding and sequence comparison to identify unusual frameworkresidues at particular positions. (See, e.g., Queen et al., U.S. Pat.No. 5,585,089; U.S. Publication Nos. 2004/0049014 and 2003/0229208; U.S.Pat. Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and5,530,101 and Riechmann et al. (1988) “Reshaping Human Antibodies ForTherapy,” Nature 332:323-327, all of which are incorporated herein byreference in their entireties.).

In a most preferred embodiment, the humanized binding domainspecifically binds to the same epitope as the donor murine antibody. Itwill be appreciated by one skilled in the art that the inventionencompasses CDR grafting of antibodies in general. Thus, the donor andacceptor antibodies may be derived from animals of the same species andeven same antibody class or sub-class. More usually, however, the donorand acceptor antibodies are derived from animals of different species.Typically the donor antibody is a non-human antibody, such as a rodentmAb, and the acceptor antibody is a human antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

The diabodies used in the methods of the invention include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the diabody. For example, but not by way of limitation, thediabody derivatives include diabodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison (1985)“Transfectomas Provide Novel Chimeric Antibodies,” Science229:1202-1207; Oi et al. (1986) “Chimeric Antibodies,” BioTechniques4:214-221; Gillies et al. (1989) “High-Level Expression Of ChimericAntibodies Using Adapted cDNA Variable Region Cassettes,” J. Immunol.Methods 125:191-202; and U.S. Pat. Nos. 6,311,415, 5,807,715, 4,816,567,and 4,816,397, which are incorporated herein by reference in theirentirety.

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; and Riechmann et al. (1988) “Reshaping HumanAntibodies For Therapy,” Nature 332:323-327, which are incorporatedherein by reference in their entireties.)

Monoclonal antibodies from which binding domains of the diabodies of theinvention can be prepared using a wide variety of techniques known inthe art including the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 0.1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones. Antigens of interest include, but are not limited to, antigensassociated with the cancers provided in section 5.8.1, antigensassociated with the autoimmune diseases and inflammatory diseasesprovided in section 5.8.2, antigens associated with the infectiousdiseases provided in section 5.8.3, and the toxins provided in section5.8.4.

Antibodies can also be generated using various phage display methodsknown in the art. In phage display methods, functional antibody domainsare displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In a particular embodiment, suchphage can be utilized to display antigen binding domains, such as Faband Fv or disulfide-bond stabilized Fv, expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage, including fd and M13. Theantigen binding domains are expressed as a recombinantly fused proteinto either the phage gene III or gene VIII protein. Examples of phagedisplay methods that can be used to make the immunoglobulins, orfragments thereof, of the present invention include those disclosed inBrinkmann et al. (1995) “Phage Display Of Disulfide-Stabilized FvFragments,” J. Immunol. Methods, 182:41-50; Ames et al. (1995)“Conversion Of Murine Fabs Isolated From A Combinatorial Phage DisplayLibrary To Full Length Immunoglobulins,” J. Immunol. Methods,184:177-186; Kettleborough et al. (1994) “Isolation Of TumorCell-Specific Single-Chain Fv From Immunized Mice Using Phage-AntibodyLibraries And The Re-Construction Of Whole Antibodies From TheseAntibody Fragments,” Eur. J. Immunol., 24:952-958; Persic et al. (1997)“An Integrated Vector System For The Eukaryotic Expression Of AntibodiesOr Their Fragments After Selection From Phage Display Libraries,” Gene,187:9-18; Burton et al. (1994) “Human Antibodies From CombinatorialLibraries,” Advances in Immunology, 57:191-280; PCT Application No.PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Phage display technology can be used to increase the affinity of anantibody for its antigen. This technique would be useful in obtaininghigh affinity antibodies. The technology, referred to as affinitymaturation, employs mutagenesis or CDR walking and re-selection usingthe cognate antigen to identify antibodies that bind with higheraffinity to the antigen when compared with the initial or parentalantibody (See, e.g., Glaser et al. (1992) “Dissection Of The CombiningSite In A Humanized Anti-Tac Antibody,” J. Immunology 149:2607-2614).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased binding affinity for the antigen can bescreened by contacting the immobilized mutants with labeled antigen. Anyscreening method known in the art can be used to identify mutantantibodies with increased avidity to the antigen (e.g., ELISA) (See Wuet al. (1998) “Stepwise In Vitro Affinity Maturation Of Vitaxin, AnAlphav Beta3-Specific Humanized mAb,” Proc Natl. Acad. Sci. USA95:6037-6042; Yelton et al. (1995) “Affinity Maturation Of The Br96Anti-Carcinoma Antibody By Codon-Based Mutagenesis,” J. Immunology155:1994-2004). CDR walking which randomizes the light chain is alsopossible (See Schier et al. (1996) “Isolation Of Picomolar AffinityAnti-C-ErbB-2 Single-Chain Fv By Molecular Evolution Of TheComplementarity Determining Regions In The Center Of The AntibodyBinding Site,” J. Mol. Bio. 263:551-567).

The present invention also encompasses the use of binding domainscomprising the amino acid sequence of any of the binding domainsdescribed herein or known in the art with mutations (e.g., one or moreamino acid substitutions) in the framework or CDR regions. Preferably,mutations in these binding domains maintain or enhance the avidityand/or affinity of the binding domains for FcγRIIB to which theyimmunospecifically bind. Standard techniques known to those skilled inthe art (e.g., immunoassays) can be used to assay the affinity of anantibody for a particular antigen.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

5.1.1 Diabodies Comprising Eptiope Binding Sites whichImmunospecifically Bind FcγRIIB

In a particular embodiment, at least one of the binding domains of thediabodies of the invention agonizes at least one activity of FcγRIIB. Inone embodiment of the invention, said activity is inhibition of B cellreceptor-mediated signaling. In another embodiment, the binding domaininhibits activation of B cells, B cell proliferation, antibodyproduction, intracellular calcium influx of B cells, cell cycleprogression, or activity of one or more downstream signaling moleculesin the FcγRIIB signal transduction pathway. In yet another embodiment,the binding domain enhances phosphorylation of FcγRIIB or SHIPrecruitment. In a further embodiment of the invention, the bindingdomain inhibits MAP kinase activity or Akt recruitment in the B cellreceptor-mediated signaling pathway. In another embodiment, the bindingdomain agonizes FcγRIIB-mediated inhibition of FcεRI signaling. In aparticular embodiment, said binding domain inhibits FcεRI-induced mastcell activation, calcium mobilization, degranulation, cytokineproduction, or serotonin release. In another embodiment, the bindingdomains of the invention stimulate phosphorylation of FcγRIIB, stimulaterecruitment of SHIP, stimulate SHIP phosphorylation and its associationwith Shc, or inhibit activation of MAP kinase family members (e.g.,Erk1, Erk2, JNK, p38, etc.). In yet another embodiment, the bindingdomains of the invention enhance tyrosine phosphorylation of p62dok andits association with SHIP and rasGAP. In another embodiment, the bindingdomains of the invention inhibit FcγR-mediated phagocytosis in monocytesor macrophages.

In another embodiment, the binding domains antagonize at least oneactivity of FcγRIIB. In one embodiment, said activity is activation of Bcell receptor-mediated signaling. In a particular embodiment, thebinding domains enhance B cell activity, B cell proliferation, antibodyproduction, intracellular calcium influx, or activity of one or moredownstream signaling molecules in the FcγRIIB signal transductionpathway. In yet another particular embodiment, the binding domainsdecrease phosphorylation of FcγRIIB or SHIP recruitment. In a furtherembodiment of the invention, the binding domains enhance MAP kinaseactivity or Akt recruitment in the B cell receptor mediated signalingpathway. In another embodiment, the binding domains antagonizeFcγRIIB-mediated inhibition of FcεRI signaling. In a particularembodiment, the binding domains enhance FcεRI-induced mast cellactivation, calcium mobilization, degranulation, cytokine production, orserotonin release. In another embodiment, the binding domains inhibitphosphorylation of FcγRIIB, inhibit recruitment of SHIP, inhibit SHIPphosphorylation and its association with Shc, enhance activation of MAPkinase family members (e.g., Erk1, Erk2, JNK, p38, etc.). In yet anotherembodiment, the binding domains inhibit tyrosine phosphorylation ofp62dok and its association with SHIP and rasGAP. In another embodiment,the binding domains enhance FcγR-mediated phagocytosis in monocytes ormacrophages. In another embodiment, the binding domains preventphagocytosis, clearance of opsonized particles by splenic macrophages.

In other embodiments, at least one of the binding domains can be used totarget the diabodies of the invention to cells that express FcγRIIB.

In one particular embodiment, one of the binding domains is derived froma mouse monoclonal antibody produced by clone 2B6 or 3H7, having ATCCaccession numbers PTA-4591 and PTA-4592, respectively. Hybridomasproducing antibodies 2B6 and 3H7 have been deposited with the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on Aug. 13, 2002 under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedures, and assigned accession numbersPTA-4591 (for hybridoma producing 2B6) and PTA-4592 (for hybridomaproducing 3H7), respectively, and are incorporated herein by reference.In a preferred embodiment, the binding domains are human or have beenhumanized, preferably are derived from a humanized version of theantibody produced by clone 3H7 or 2B6.

The invention also encompasses diabodies with binding domains from otherantibodies, that specifically bind FcγRIIB, preferably human FcγRIIB,more preferably native human FcγRIIB, that are derived from clonesincluding but not limited to 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCCAccession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively. Hybridomas producing the above-identified clones weredeposited under the provisions of the Budapest Treaty With the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on May 7, 2004, and are incorporated herein by reference. Inpreferred embodiments, the binding domains from the antibodies describedabove are humanized.

In a specific embodiment, the binding domains used in the diabodies ofthe present invention are from an antibody or an antigen-bindingfragment thereof (e.g., comprising one or more complementarilydetermining regions (CDRs), preferably all 6 CDRs) of the antibodyproduced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In anotherembodiment, the binding domain binds to the same epitope as the mousemonoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2, respectively and/or competes with the mouse monoclonal antibodyproduced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 as determined,e.g., in an ELISA assay or other appropriate competitive immunoassay,and also binds FcγRIIB with a greater affinity than the binding domainbinds FcγRIIA.

The present invention also encompasses diabodies with binding domainscomprising an amino acid sequence of a variable heavy chain and/orvariable light chain that is at least 45%, at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical to theamino acid sequence of the variable heavy chain and/or light chain ofthe mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2. The present invention further encompasses diabodies withbinding domains that specifically bind FcγRIIB with greater affinitythan said antibody or fragment thereof binds FcγRIIA, and that comprisean amino acid sequence of one or more CDRs that is at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the amino acid sequence of one or more CDRs ofthe mouse monoclonal antibody produced by clone 2B6, 31-17, 1 D5, 2E1,21-19, 2D11, or 1F2. The determination of percent identity of two aminoacid sequences can be determined by any method known to one skilled inthe art, including BLAST protein searches.

The present invention also encompasses the use of diabodies containingbinding domains that specifically bind FcγRIIB with greater affinitythan binding domain binds FcγRIIA, which are encoded by a nucleotidesequence that hybridizes to the nucleotide sequence of the mousemonoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or1F2 under stringent conditions. In a preferred embodiment, the bindingdomain specifically binds FcγRIIB with greater affinity than FcγRIIA,and comprises a variable light chain and/or variable heavy chain encodedby a nucleotide sequence that hybridizes under stringent conditions tothe nucleotide sequence of the variable light chain and/or variableheavy chain of the mouse monoclonal antibody produced by clone 2B6, 3H7,1D5, 2E1, 2H9, 2D11, or 1F2 under stringent conditions. In anotherpreferred embodiment, the binding domains specifically bind FcγRIIB withgreater affinity than FcγRIIA, and comprise one or more CDRs encoded bya nucleotide sequence that hybridizes under stringent conditions to thenucleotide sequence of one or more CDRs of the mouse monoclonal antibodyproduced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. Stringenthybridization conditions include, but are not limited to, hybridizationto filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1×SSC/0.2% SDS at about 60° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3, incorporated hereinby reference).

The present invention also encompasses the use of binding domainscomprising the amino acid sequence of any of the binding domainsdescribed above with mutations (e.g., one or more amino acidsubstitutions) in the framework or CDR regions. Preferably, mutations inthese binding domains maintain or enhance the avidity and/or affinity ofthe binding domains for FcγRIIB to which they immunospecifically bind.Standard techniques known to those skilled in the art (e.g.,immunoassays) can be used to assay the affinity of an antibody for aparticular antigen.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

In preferred embodiments, the binding domains are derived from humanizedantibodies. A humanized FcγRIIB specific antibody may comprisesubstantially all of at least one, and typically two, variable domainsin which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence.

The diabodies of present invention comprise humanized variable domainsspecific for FcγRIIB in which one or more regions of one or more CDRs ofthe heavy and/or light chain variable regions of a human antibody (therecipient antibody) have been substituted by analogous parts of one ormore CDRs of a donor monoclonal antibody which specifically bindsFcγRIIB, with a greater affinity than FcγRIIA, e.g., a monoclonalantibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. Inother embodiments, the humanized antibodies bind to the same epitope as2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, respectively.

In a preferred embodiment, the CDR regions of the humanized FcγRIIBbinding domain are derived from a murine antibody specific for FcγRIIB.In some embodiments, the humanized antibodies described herein comprisealterations, including but not limited to amino acid deletions,insertions, modifications, of the acceptor antibody, i.e., human, heavyand/or light chain variable domain framework regions that are necessaryfor retaining binding specificity of the donor monoclonal antibody. Insome embodiments, the framework regions of the humanized antibodiesdescribed herein does not necessarily consist of the precise amino acidsequence of the framework region of a natural occurring human antibodyvariable region, but contains various alterations, including but notlimited to amino acid deletions, insertions, modifications that alterthe property of the humanized antibody, for example, improve the bindingproperties of a humanized antibody region that is specific for the sametarget as the murine FcγRIIB specific antibody. In most preferredembodiments, a minimal number of alterations are made to the frameworkregion in order to avoid large-scale introductions of non-humanframework residues and to ensure minimal immunogenicity of the humanizedantibody in humans. The donor monoclonal antibody is preferably amonoclonal antibody produced by clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or1F2.

In a specific embodiment, the binding domain encompasses variabledomains of a CDR-grafted antibody which specifically binds FcγRIIB witha greater affinity than said antibody binds FcγRIIA, wherein theCDR-grafted antibody comprises a heavy chain variable region domaincomprising framework residues of the recipient antibody and residuesfrom the donor monoclonal antibody, which specifically binds FcγRIIBwith a greater affinity than said antibody binds FcγRIIA, e.g.,monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2. In another specific embodiment, the diabodies of the inventioncomprise variable domains from a CDR-grafted antibody which specificallybinds FcγRIIB with a greater affinity than said antibody binds FcγRIIA,wherein the CDR-grafted antibody comprises a light chain variable regiondomain comprising framework residues of the recipient antibody andresidues from the donor monoclonal antibody, which specifically bindsFcγRIIB with a greater affinity than said antibody binds FcγRIIA, e.g.,monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2.

The humanized anti-FcγRIIB variable domains used in the invention mayhave a heavy chain variable region comprising the amino acid sequence ofCDR1 (SEQ ID NO: 24 or SEQ ID NO: 25) and/or CDR2 (SEQ ID NO: 26 or SEQID NO: 27) and/or CDR3 (SEQ ID NO: 28 or SEQ ID NO: 29) and/or a lightchain variable region comprising the amino acid sequence of CDR1 (SEQ IDNO: 30 or SEQ ID NO: 31) and/or a CDR2 (SEQ ID NO: 32, SEQ ID NO: 33,SEQ ID NO: 34, or SEQ ID NO: 35) and/or CDR3 (SEQ ID NO: 36 or SEQ IDNO: 37).

In one specific embodiment, the diabody comprises variable domains froma humanized 2B6 antibody, wherein the VH region consists of the FRsegments from the human germline VH segment VH1-18 (Matsuda et al.(1998) “The Complete Nucleotide Sequence Of The Human ImmunoglobulinHeavy Chain Variable Region Locus,” J. Exp. Med. 188:2151-2162) and JH6(Ravetch et al. (1981) “Structure Of The Human Immunoglobulin Mu Locus:Characterization Of Embryonic And Rearranged J And D Genes,” Cell 27(3Pt. 2): 583-91), and one or more CDR regions of the 2B6 VH, having theamino acid sequence of SEQ ID NO:24, SEQ ID NO: 26, or SEQ ID NO: 28. Inone embodiment, the 2B6 VH has the amino acid sequence of SEQ ID NO: 38.In another embodiment the 2B6 VH domain has the amino acid sequence ofHu2B6VH, SEQ ID NO: 85, and can be encoded by the nucleotide sequence ofSEQ ID NO: 86. In another specific embodiment, the diabody furthercomprises a VL region, which consists of the FR segments of the humangermline VL segment VK-A26 (Lautner-Rieske et al. (1992) “The HumanImmunoglobulin Kappa Locus. Characterization Of The Duplicated ARegions,” Eur. J. Immunol. 22:1023-1029) and JK4 (Hieter et al. (1982)“Evolution Of Human Immunoglobulin Kappa J Region Genes,” J. Biol. Chem.257:1516-22), and one or more CDR regions of 2B6VL, having the aminoacid sequence of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, and SEQ ID NO: 36. In one embodiment, the 2B6 VL has the amino acidsequence of SEQ ID NO: 39; SEQ ID NO: 40, or SEQ ID NO: 41. In aspecific embodiment, the 2B6 VL has the amino acid sequence of Hu2B6VL,SEQ ID NO: 87, and can be encoded by the nucleotide sequence provided inSEQ ID NO: 88.

In another specific embodiment, the diabody has variable domains from ahumanized 3H7 antibody, wherein the VH region consists of the FRsegments from a human germline VH segment and the CDR regions of the 3H7VH, having the amino acid sequence of SEQ ID NO. 35. In another specificembodiment, the humanized 3H7 antibody further comprises a VL regions,which consists of the FR segments of a human germline VL segment and theCDR regions of 31-17VL, having the amino acid sequence of SEQ ID NO: 42.

In particular, binding domains immunospecifically bind to extracellulardomains of native human FcγRIIB, and comprise (or alternatively, consistof) CDR sequences of 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, in any ofthe following combinations: a VH CDR1 and a VL CDR1; a VH CDR1 and a VLCDR2; a VH CDR1 and a VL CDR3; a VH CDR2 and a VL CDR1; VH CDR2 and VLCDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VH CDR1; a VH CDR3 and aVL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, a VH CDR2 and a VL CDR1; aVH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, a VH CDR2 and a VL CDR3; aVH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; aVH CDR2, a VH CDR2 and a VL CDR3; a VH CDR1, a VL CDR1 and a VL CDR2; aVH CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, a VL CDR1 and a VL CDR2; aVH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, a VL CDR1 and a VL CDR2; aVH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3 and aVL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR1, a VHCDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR3, a VL CDR1 and a VLCDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR2 and a VLCDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR1, a VH CDR3, a VLCDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1, a VLCDR2, and a VL CDR3; or any combination thereof of the VH CDRs and VLCDRs disclosed herein.

Antibodies for deriving binding domains to be included in the diabodiesof the invention may be further characterized by epitope mapping, sothat antibodies may be selected that have the greatest specificity forFcγRIIB compared to FcγRIIA. Epitope mapping methods of antibodies arewell known in the art and encompassed within the methods of theinvention. In certain embodiments fusion proteins comprising one or moreregions of FcγRIIB may be used in mapping the epitope of an antibody ofthe invention. In a specific embodiment, the fusion protein contains theamino acid sequence of a region of an FcγRIIB fused to the Fc portion ofhuman IgG2. Each fusion protein may further comprise amino acidsubstitutions and/or replacements of certain regions of the receptorwith the corresponding region from a homolog receptor, e.g., FcγRIIA, asshown in Table 2 below. pMGX125 and pMGX132 contain the IgG binding siteof the FcγRIIB receptor, the former with the C-terminus of FcγRIIB andthe latter with the C-terminus of FcγRIIA and can be used todifferentiate C-terminus binding. The others have FcγRIIA substitutionsin the IgG binding site and either the FcγIIA or FcγIIB N-terminus.These molecules can help determine the part of the receptor moleculewhere the antibodies bind.

TABLE 2 List of the fusion proteins that may be used to investigate theepitope of the monoclonal anti-FcγRIIB antibodies. Residues 172 to 180belong to the IgG binding site of FcγRIIA and B. The specific aminoacids from FcγRIIA sequence are in bold. SEQ ID Plasmid ReceptorN-terminus 172-180 NO: C-terminus pMGX125 RIIb IIb KKFSRSDPN 43APS------SS (IIb) pMGX126 RIIa/b IIa QKFSRLDPN 44 APS------SS (IIb)pMGX127 IIa QKFSRLDPT 45 APS------SS (IIb) pMGX128 IIb KKFSRLDPT 46APS------SS (IIb) pMGX129 IIa QKFSHLDPT 47 APS------SS (IIb) pMGX130 IIbKKFSHLDPT 48 APS------SS (IIb) pMGX131 IIa QKFSRLDPN 49 VPSMGSSS(IIa)pMGX132 IIb KKFSRSDPN 50 VPSMGSSS(IIa) pMGX133 RIIa-131R IIa QKFSRLDPT51 VPSMGSSS(IIa) pMGX134 RIIa-131H IIa QKFSHLDPT 52 VPSMGSSS(IIa)pMGX135 IIb KKFSRLDPT 53 VPSMGSSS(IIa) pMGX136 IIb KKFSHLDPT 54VPSMGSSS(IIa) Note: APSSS is SEQ ID NO: 309; VPSMGSSS is SEQ ID NO: 310

The fusion proteins may be used in any biochemical assay fordetermination of binding to an anti-FcγRIIB antibody of the invention,e.g., an ELISA. In other embodiments, further confirmation of theepitope specificity may be done by using peptides with specific residuesreplaced with those from the FcγRIIA sequence.

The antibodies can be characterized using assays for identifying thefunction of the antibodies of the invention, particularly the activityto modulate FcγRIIB signaling. For example, characterization assays ofthe invention can measure phosphorylation of tyrosine residues in theITIM motif of FcγRIIB, or measure the inhibition of B cellreceptor-generated calcium mobilization. The characterization assays ofthe invention can be cell-based or cell-free assays.

It has been well established in the art that in mast cells coaggregationof FcγRIIB with the high affinity IgE receptor, FcεRI, leads toinhibition of antigen-induced degranulation, calcium mobilization, andcytokine production (Metcalfe D. D. et al. (1997) “Mast Cells,” Physiol.Rev. 77:1033-1079; Long E. O. (1999) “Regulation Of Immune ResponsesThrough Inhibitory Receptors,” Annu. Rev. Immunol. 17: 875-904). Themolecular details of this signaling pathway have been recentlyelucidated (Ott V. L. (2002) “Downstream Of Kinase, p62(dok), Is AMediator Of FcgammaIIB Inhibition Of Fc Epsilon RI Signaling,” J.Immunol. 162(9):4430-4439). Once coaggregated with FcεRI, FcγRIIB israpidly phosphorylated on tyrosine in its ITIM motif, and then recruitsSrc Homology-2 containing inositol-5-phosphatase (SHIP), an SH2domain-containing inosital polyphosphate 5-phosphatase, which is in turnphosphorylated and associates with Shc and p62^(dok) (p62^(dok) is theprototype of a family of adaptor molecules, which includes signalingdomains such as an aminoterminal pleckstrin homology domain (PH domain),a PTB domain, and a carboxy terminal region containing PXXP motifs andnumerous phosphorylation sites (Carpino et al. (1997) “p62(dok): AConstitutively Tyrosine-Phosphorylated, GAP-Associated Protein InChronic Myelogenous Leukemia Progenitor Cells,” Cell, 88: 197-204;Yamanshi et al. (1997) “Identification Of The Abl- And rasGAP-Associated62 kDa Protein As A Docking Protein, Dok,” Cell, 88:205-211).

The anti-FcγRIIB antibodies for use in the invention may likewise becharacterized for ability to modulate one or more IgE mediatedresponses. Preferably, cells lines co-expressing the high affinityreceptor for IgE and the low affinity receptor for FcγRIIB will be usedin characterizing the anti-FcγRIIB antibodies in modulating IgE mediatedresponses. In a specific embodiment, cells from a rat basophilicleukemia cell line (RBL-H23; Barsumian E. L. et al. (1981) “IgE-InducedHistamine Release From Rat Basophilic Leukemia Cell Lines: Isolation OfReleasing And Nonreleasing Clones,” Eur. J. Immunol. 11:317-323, whichis incorporated herein by reference in its entirety) transfected withfull length human FcγRIIB will be used. RBL-2H3 is a well characterizedrat cell line that has been used extensively to study the signalingmechanisms following IgE-mediated cell activation. When expressed inRBL-2H3 cells and coaggregated with FcεRI, FcγRIIB inhibitsFcεRI-induced calcium mobilization, degranulation, and cytokineproduction (Malbec et al. (1998) “Fc Epsilon Receptor I-AssociatedLyn-Dependent Phosphorylation Of Fc Gamma Receptor IIB During NegativeRegulation Of Mast Cell Activation,” J. Immunol. 160:1647-1658; Daeronet al. (1995) “Regulation Of High-Affinity IgE Receptor-Mediated MastCell Activation By Murine Low-Affinity IgG Receptors,” J. Clin. Invest.95:577; Ott V. L. (2002) “Downstream Of Kinase, p62(dok), Is A MediatorOf FcgammaIIB Inhibition Of Fc Epsilon RI Signaling,” J. Immunol.162(9):4430-4439).

Antibodies for use in the invention may also be characterized forinhibition of FcεRI induced mast cell activation. For example, cellsfrom a rat basophilic leukemia cell line (RBL-H23; Barsumian E. L. etal. (1981) “IgE-Induced Histamine Release From Rat Basophilic LeukemiaCell Lines: Isolation Of Releasing And Nonreleasing Clones,” Eur. J.Immunol. 11:317-323) that have been transfected with FcγRIIB aresensitized with IgE and stimulated either with F(ab′)₂ fragments ofrabbit anti-mouse IgG, to aggregate FcεRI alone, or with whole rabbitanti-mouse IgG to coaggregate FcγRIIB and FcεRI. In this system,indirect modulation of down stream signaling molecules can be assayedupon addition of antibodies of the invention to the sensitized andstimulated cells. For example, tyrosine phosphorylation of FcγRIIB andrecruitment and phosphorylation of SHIP, activation of MAP kinase familymembers, including but not limited to Erk1, Erk2, JNK, or p38; andtyrosine phosphorylation of p62^(dok) and its association with SHIP andRasGAP can be assayed.

One exemplary assay for determining the inhibition of FcεRI induced mastcell activation by the antibodies of the invention can comprise of thefollowing: transfecting RBL-H23 cells with human FcγRIIB; sensitizingthe RBL-H23 cells with IgE; stimulating RBL-H23 cells with eitherF(ab′)₂ of rabbit anti-mouse IgG (to aggregate FcεRI alone and elicitFcεRI-mediated signaling, as a control), or stimulating RBL-H23 cellswith whole rabbit anti-mouse IgG to (to coaggregate FcγRIIB and FcεRI,resulting in inhibition of FcεRI-mediated signaling). Cells that havebeen stimulated with whole rabbit anti-mouse IgG antibodies can befurther pre-incubated with the antibodies of the invention. MeasuringFcεRI-dependent activity of cells that have been pre-incubated with theantibodies of the invention and cells that have not been pre-incubatedwith the antibodies of the invention, and comparing levels ofFcεRI-dependent activity in these cells, would indicate a modulation ofFcεRI-dependent activity by the antibodies of the invention.

The exemplary assay described above can be for example, used to identifyantibodies that block ligand (IgG) binding to FcγRIIB receptor andantagonize FcγRIIB-mediated inhibition of FcεRI signaling by preventingcoaggregating of FcγRIIB and FcεRI. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcεRI and agonizeFcγRIIB-mediated inhibition of FcεRI signaling by promotingcoaggregating of FcγRIIB and FcεRI.

In some embodiments, the anti-FcγRIIB diabodies, comprising the epitopebinding domains of anti-FcγRIIB antibodies identified described hereinor known in the art, of the invention are characterized for theirability to modulate an IgE mediated response by monitoring and/ormeasuring degranulation of mast cells or basophils, preferably in acell-based assay. Preferably, mast cells or basophils for use in suchassays have been engineered to contain human FcγRIIB using standardrecombinant methods known to one skilled in the art. In a specificembodiment the anti-FcγRIIB antibodies of the invention arecharacterized for their ability to modulate an IgE mediated response ina cell-based β-hexosaminidase (enzyme contained in the granules) releaseassay. β-hexosaminidase release from mast cells and basophils is aprimary event in acute allergic and inflammatory condition (Aketani etal. (2001) “Correlation Between Cytosolic Calcium Concentration AndDegranulation In RBL-2H3 Cells In The Presence Of Various ConcentrationsOf Antigen-Specific IgEs,” Immunol. Lett. 75: 185-189; Aketani et al.(2000) “A Screening Method For Antigen-Specific IgE Using Mast CellsBased On Intracellular Calcium Signaling,” Anal. Chem. 72: 2653-2658).Release of other inflammatory mediators including but not limited toserotonin and histamine may be assayed to measure an IgE mediatedresponse in accordance with the methods of the invention. Although notintending to be bound by a particular mechanism of action, release ofgranules such as those containing β-hexosaminidase from mast cells andbasophils is an intracellular calcium concentration dependent processthat is initiated by the cross-linking of FcγRIs with multivalentantigen.

The ability to study human mast cells has been limited by the absence ofsuitable long term human mast cell cultures. Recently two novel stemcell factor dependent human mast cell lines, designated LAD 1 and LAD2,were established from bone marrow aspirates from a patient with mastcell sarcoma/leukemia (Kirshenbaum et al. (2003) “Characterization OfNovel Stem Cell Factor Responsive Human Mast Cell Lines LAD 1 And 2Established From A Patient With Mast Cell Sarcoma/Leukemia; ActivationFollowing Aggregation Of FcRI Or FcγRI,” Leukemia research, 27:677-82,which is incorporated herein by reference in its entirety.). Both celllines have been described to express FcεR1 and several human mast cellmarkers. LAD 1 and 2 cells can be used for assessing the effect of theantibodies of the invention on IgE mediated responses. In a specificembodiment, cell-based β-hexosaminidase release assays such as thosedescribed supra may be used in LAD cells to determine any modulation ofthe IgE-mediated response by the anti-FcγRIIB antibodies of theinvention. In an exemplary assay, human mast cells, e.g., LAD 1, areprimed with chimeric human IgE anti-nitrophenol (NP) and challenged withBSA-NP, the polyvalent antigen, and cell degranulation is monitored bymeasuring the β-hexosaminidase released in the supernatant (Kirshenbaumet al. (2003) “Characterization Of Novel Stem Cell Factor ResponsiveHuman Mast Cell Lines LAD 1 And 2 Established From A Patient With MastCell Sarcoma/Leukemia; Activation Following Aggregation Of FcRI OrFcγRI,” Leukemia research, 27:677-82, which is incorporated herein byreference in its entirety.).

In some embodiments, if human mast cells have a low expression ofendogenous FcγRIIB, as determined using standard methods known in theart, e.g., FACS staining, it may be difficult to monitor and/or detectdifferences in the activation of the inhibitory pathway mediated by theanti-FcγRIIB diabodies of the invention. The invention thus encompassesalternative methods, whereby the FcγRIIB expression may be upregulatedusing cytokines and particular growth conditions. FcγRIIB has beendescribed to be highly up-regulated in human monocyte cell lines, e.g.,THP1 and U937, (Tridandapani et al. (2002) “Regulated Expression AndInhibitory Function Of Fcgamma RIIB In Human Monocytic Cells,” J. Biol.Chem., 277(7): 5082-5089) and in primary human monocytes (Pricop et al.(2001) “Differential Modulation Of Stimulatory And Inhibitory Fc GammaReceptors On Human Monocytes By Th1 And Th2 Cytokines,” J. of Immunol.,166: 531-537) by IL4. Differentiation of U937 cells with dibutyrylcyclic AMP has been described to increase expression of FcγRII (Cameronet al. (2002) “Differentiation Of The Human Monocyte Cell Line, U937,With Dibutyryl CyclicAMP Induces The Expression Of The Inhibitory FcReceptor, FcgammaRIIB,” Immunology Letters 83, 171-179). Thus theendogenous FcγRIIB expression in human mast cells for use in the methodsof the invention may be up-regulated using cytokines, e.g., IL-4, IL-13,in order to enhance sensitivity of detection.

The anti-FcγRIIB diabodies can also be assayed for inhibition of B-cellreceptor (BCR)-mediated signaling. BCR-mediated signaling can include atleast one or more down stream biological responses, such as activationand proliferation of B cells, antibody production, etc. Coaggregation ofFcγRIIB and BCR leads to inhibition of cell cycle progression andcellular survival. Further, coaggregation of FcγRIIB and BCR leads toinhibition of BCR-mediated signaling.

Specifically, BCR-mediated signaling comprises at least one or more ofthe following: modulation of down stream signaling molecules (e.g.,phosphorylation state of FcγRIIB, SHIP recruitment, localization of Btkand/or PLCγ, MAP kinase activity, recruitment of Akt (anti-apoptoticsignal), calcium mobilization, cell cycle progression, and cellproliferation.

Although numerous effector functions of FcγRIIB-mediated inhibition ofBCR signaling are mediated through SHIP, recently it has beendemonstrated that lipopolysaccharide (LPS)-activated B cells from SHIPdeficient mice exhibit significant FcγRIIB-mediated inhibition ofcalcium mobilization, Ins(1,4,5)P₃ production, and Erk and Aktphosphorylation (Brauweiler et al. (2001) “Partially Distinct MolecularMechanisms Mediate Inhibitory FcgammaRIIB Signaling In Resting AndActivated B Cells,” Journal of Immunology, 167(1): 204-211).Accordingly, ex vivo B cells from SHIP deficient mice can be used tocharacterize the antibodies of the invention. One exemplary assay fordetermining FcγRIIB-mediated inhibition of BCR signaling by theantibodies of the invention can comprise the following: isolatingsplenic B cells from SHIP deficient mice, activating said cells withlipopolysachharide, and stimulating said cells with either F(ab′)₂anti-IgM to aggregate BCR or with anti-IgM to coaagregate BCR withFcγRIIB. Cells that have been stimulated with intact anti-IgM tocoaggregate BCR with FcγRIIB can be further pre-incubated with theantibodies of the invention. FcγRIIB-dependent activity of cells can bemeasured by standard techniques known in the art. Comparing the level ofFcγRIIB-dependent activity in cells that have been pre-incubated withthe antibodies and cells that have not been pre-incubated, and comparingthe levels would indicate a modulation of FcγRIIB-dependent activity bythe antibodies.

Measuring FcγRIIB-dependent activity can include, for example, measuringintracellular calcium mobilization by flow cytometry, measuringphosphorylation of Akt and/or Erk, measuring BCR-mediated accumulationof PI(3,4,5)P₃, or measuring FcγRIIB-mediated proliferation B cells.

The assays can be used, for example, to identify diabodies oranti-FcγRIIB antibodies for use in the invention that modulateFcγRIIB-mediated inhibition of BCR signaling by blocking the ligand(IgG) binding site to FcγRIIB receptor and antagonizing FcγRIIB-mediatedinhibition of BCR signaling by preventing coaggregation of FcγRIIB andBCR. The assays can also be used to identify antibodies that enhancecoaggregation of FcγRIIB and BCR and agonize FcγRIIB-mediated inhibitionof BCR signaling.

The anti-FcγRIIB antibodies can also be assayed for FcγRII-mediatedsignaling in human monocytes/macrophages. Coaggregation of FcγRIIB witha receptor bearing the immunoreceptor tyrosine-based activation motif(ITAM) acts to down-regulate FcγR-mediated phagocytosis using SHIP asits effector (Tridandapani et al. (2002) “Regulated Expression AndInhibitory Function Of Fcgamma RIIB In Human Monocytic Cells,” J. Biol.Chem., 277(7): 5082-5089). Coaggregation of FcγRIIA with FcγRIIB resultsin rapid phosphorylation of the tyrosine residue on FcγRIIB's ITIMmotif, leading to an enhancement in phosphorylation of SHIP, associationof SHIP with Shc, and phosphorylation of proteins having the molecularweight of 120 and 60-65 kDa. In addition, coaggregation of FcγRIIA withFcγRIIB results in down-regulation of phosphorylation of Akt, which is aserine-threonine kinase that is involved in cellular regulation andserves to suppress apoptosis.

The anti-FcγRIIB diabodies can also be assayed for inhibition ofFcγR-mediated phagocytosis in human monocytes/macrophages. For example,cells from a human monocytic cell line, TI-IP-1 can be stimulated eitherwith Fab fragments of mouse monoclonal antibody IV.3 against FcγRII andgoat anti-mouse antibody (to aggregate FcγRIIA alone), or with wholeIV.3 mouse monoclonal antibody and goat anti-mouse antibody (tocoaggregate FcγRIIA and FcγRIIB). In this system, modulation of downstream signaling molecules, such as tyrosine phosphorylation of FcγRIIB,phosphorylation of SHIP, association of SHIP with Shc, phosphorylationof Akt, and phosphorylation of proteins having the molecular weight of120 and 60-65 kDa can be assayed upon addition of molecules of theinvention to the stimulated cells. In addition, FcγRIIB-dependentphagocytic efficiency of the monocyte cell line can be directly measuredin the presence and absence of the antibodies of the invention.

Another exemplary assay for determining inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages by the antibodies of theinvention can comprise the following: stimulating THP-1 cells witheither Fab of IV.3 mouse anti-FcγRII antibody and goat anti-mouseantibody (to aggregate FcγRIIA alone and elicit FcγRIIA-mediatedsignaling); or with mouse anti-FcγRII antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB and inhibitingFcγRIIA-mediated signaling. Cells that have been stimulated with mouseanti-FcγRII antibody and goat anti-mouse antibody can be furtherpre-incubated with the molecules of the invention. MeasuringFcγRIIA-dependent activity of stimulated cells that have beenpre-incubated with molecules of the invention and cells that have notbeen pre-incubated with the antibodies of the invention and comparinglevels of FcγRIIA-dependent activity in these cells would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention.

The exemplary assay described can be used for example, to identifybinding domains that block ligand binding of FcγRIIB receptor andantagonize FcγRIIB-mediated inhibition of FcγRIIA signaling bypreventing coaggregation of FcγRIIB and FcγRIIA. This assay likewiseidentifies binding domains that enhance coaggregation of FcγRIIB andFcγRIIA and agonize FcγRIIB-mediated inhibition of FcγRIIA signaling.

The FcγRIIB binding domains of interest can be assayed while comprised Iantibodies by measuring the ability of THP-1 cells to phagocytosefluoresceinated IgG-opsonized sheep red blood cells (SRBC) by methodspreviously described (Tridandapani et al. (2000) “The Adapter ProteinLAT Enhances Fcgamma Receptor-Mediated Signal Transduction In MyeloidCells,” J. Biol. Chem. 275: 20480-7). For example, an exemplary assayfor measuring phagocytosis comprises of: treating THP-1 cells with theantibodies of the invention or with a control antibody that does notbind to FcγRII, comparing the activity levels of said cells, wherein adifference in the activities of the cells (e.g., rosetting activity (thenumber of THP-1 cells binding IgG-coated SRBC), adherence activity (thetotal number of SRBC bound to THP-1 cells), and phagocytic rate) wouldindicate a modulation of FcγRIIA-dependent activity by the antibodies ofthe invention. This assay can be used to identify, for example,antibodies that block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of phagocytosis. This assay can alsoidentify antibodies that enhance FcγRIIB-mediated inhibition of FcγRIIAsignaling.

In a preferred embodiment, the binding domains modulateFcγRIIB-dependent activity in human monocytes/macrophages in at leastone or more of the following ways: modulation of downstream signalingmolecules (e.g., modulation of phosphorylation state of FcγRIIB,modulation of SHIP phosphorylation, modulation of SHIP and Shcassociation, modulation of phosphorylation of Akt, modulation ofphosphorylation of additional proteins around 120 and 60-65 kDa) andmodulation of phagocytosis.

5.1.2 CD16A Binding Domains

The following section discusses CD16A binding proteins which can be usedas sources for light and heavy chain variable regions for covalentdiabody production. In the present invention CD16A binding proteinsincludes molecules comprising VL and VH domains of anti-CD16Aantibodies, which VH and VL domains are used in the production of thediabodies of the present invention.

A variety of CD16A binding proteins may be used in connection with thepresent invention. Suitable CD16A binding proteins include human orhumanized monoclonal antibodies as well as CD16A binding antibodyfragments (e.g., scFv or single chain antibodies, Fab fragments,minibodies) and another antibody-like proteins that bind to CD16A via aninteraction with a light chain variable region domain, a heavy chainvariable region domain; or both.

In some embodiments, the CD16A binding protein for use according to theinvention comprises a VL and/or VH domain that has one or more CDRs withsequences derived from a non-human anti-CD16A antibody, such as a mouseanti-CD16A antibody, and one or more framework regions with derived fromframework sequences of one or more human immunoglobulins. A number ofnon-human anti-CD16A monoclonal antibodies, from which CDR and othersequences may be obtained, are known (see, e.g., Tamm et al. (1996) “TheBinding Epitopes Of Human CD16 (Fc gamma RIII) Monoclonal Antibodies.Implications For Ligand Binding,” J. Imm. 157:1576-81; Fleit et al.(1989) p. 159; LEUKOCYTE TYPING II: HUMAN MYELOID AND HEMATOPOIETICCELLS, Reinherz et al., eds. New York: Springer-Verlag; (1986);LEUCOCYTE TYPING III: WHITE CELL DIFFERENTIATION ANTIGENS McMichael A J,ed., Oxford: Oxford University Press, 1986); LEUKOCYTE TYPING IV: WHITECELL DIFFERENTIATION ANTIGENS, Kapp et al., eds. Oxford Univ. Press,Oxford; LEUKOCYTE TYPING V: WHITE CELL DIFFERENTIATION ANTIGENS,Schlossman et al., eds. Oxford Univ. Press, Oxford; LEUKOCYTE TYPING VI:WHITE CELL DIFFERENTIATION ANTIGENS, Kishimoto, ed. Taylor & Francis. Inaddition, as shown in the Examples, new CD16A binding proteins thatrecognize human CD16A expressed on cells can be obtained using wellknown methods for production and selection of monoclonal antibodies orrelated binding proteins (e.g., hybridoma technology, phage display, andthe like). See, for example, O'Connell et al. (2002) “Phage VersusPhagemid Libraries For Generation Of Human Monoclonal Antibodies,” J.Mol. Biol. 321:49-56; Hoogenboom et al. (2000) “Natural And DesignerBinding Sites Made By Phage Display Technology,” Imm. Today 21:371078;Krebs et al. (2001) “High-Throughput Generation And Engineering OfRecombinant Human Antibodies,” J. Imm. Methods 254:67-84; and otherreferences cited herein. Monoclonal antibodies from a non-human speciescan be chimerized or humanized using techniques using techniques ofantibody humanization known in the art.

Alternatively, fully human antibodies against CD16A can be producedusing transgenic animals having elements of a human immune system (see,e.g., U.S. Pat. Nos. 5,569,825 and 5,545,806), using human peripheralblood cells (Casali et al. (1986) “Human Monoclonals FromAntigen-Specific Selection Of B Lymphocytes And Transformation By EBV,”Science 234:476-479), by screening a DNA library from human B cellsaccording to the general protocol outlined by Huse et al. (1989)“Generation Of A Large Combinatorial Library Of The ImmunoglobulinRepertoire In Phage Lambda,” Science 246:1275-1281, and by othermethods.

In a preferred embodiment, the binding donor is from the 3G8 antibody ora humanized version thereof, e.g., such as those disclosed in U.S.patent application publication 2004/0010124, which is incorporated byreference herein in its entirety. It is contemplated that, for somepurposes, it may be advantageous to use CD16A binding proteins that bindthe CD16A receptor at the same epitope bound by 3G8, or at leastsufficiently close to this epitope to block binding by 3G8. Methods forepitope mapping and competitive binding experiments to identify bindingproteins with the desired binding properties are well known to thoseskilled in the art of experimental immunology. See, for example, Harlowand Lane, cited supra; Stähli et al. (1983) “Distinction Of Epitopes ByMonoclonal Antibodies,” Methods in Enzymology 92:242-253; Kirkland etal. (1986) “Analysis Of The Fine Specificity And Cross-Reactivity OfMonoclonal Anti-Lipid A Antibodies,” J. Immunol. 137:3614-3619; Morel etal. (1988) “Monoclonal Antibodies To Bovine Serum Albumin: Affinity AndSpecificity Determinations,” Molec. Immunol. 25:7-15; Cheung et al.(1990) “Epitope-Specific Antibody Response To The Surface Antigen OfDuck Hepatitis B Virus In Infected Ducks,” Virology 176:546-552; andMoldenhauer et al. (1990) “Identity Of HML-1 Antigen On IntestinalIntraepithelial T Cells And Of B-ly7 Antigen On Hairy Cell Leukaemia,”Scand. J. Immunol. 32:77-82. For instance, it is possible to determineif two antibodies bind to the same site by using one of the antibodiesto capture the antigen on an ELISA plate and then measuring the abilityof the second antibody to bind to the captured antigen. Epitopecomparison can also be achieved by labeling a first antibody, directlyor indirectly, with an enzyme, radionuclide or fluorophore, andmeasuring the ability of an unlabeled second antibody to inhibit thebinding of the first antibody to the antigen on cells, in solution, oron a solid phase.

It is also possible to measure the ability of antibodies to block thebinding of the CD16A receptor to immune complexes formed on ELISAplates. Such immune complexes are formed by first coating the plate withan antigen such as fluorescein, then applying a specificanti-fluorescein antibody to the plate. This immune complex then servesas the ligand for soluble Fc receptors such as sFcRIIIa. Alternatively asoluble immune complex may be formed and labeled, directly orindirectly, with an enzyme radionuclide or fluorophore. The ability ofantibodies to inhibit the binding of these labeled immune complexes toFc receptors on cells, in solution or on a solid phase can then bemeasured.

CD16A binding proteins of the invention may or may not comprise a humanimmunoglobulin Fc region. Fc regions are not present, for example, inscFv binding proteins. Fc regions are present, for example, in human orhumanized tetrameric monoclonal IgG antibodies. As described supra, insome embodiments of the present invention, the CD16A binding proteinincludes an Fc region that has an altered effector function, e.g.,reduced affinity for an effector ligand such as an Fc receptor orC₁₋component of complement compared to the unaltered Fc region (e.g., Fcof naturally occurring IgG1, proteins). In one embodiment the Fc regionis not glycosylated at the Fc region amino acid corresponding toposition 297. Such antibodies lack Fc effector function.

Thus, the CD16A binding protein may not exhibit Fc-mediated binding toan effector ligand such as an Fc receptor or the C₁₋component ofcomplement due to the absence of the Fc domain in the binding proteinwhile, in other cases, the lack of binding or effector function is dueto an alteration in the constant region of the antibody.

5.1.2.1 CD16A Binding Proteins Comprising CDR Sequences Similar to a mAb3G8 CDR Sequences

CD16A binding proteins that can be used in the practice of the inventioninclude proteins comprising a CDR sequence derived from (i.e., having asequence the same as or similar to) the CDRs of the mouse monoclonalantibody 3G8. Complementary cDNAs encoding the heavy chain and lightchain variable regions of the mouse 3G8 monoclonal antibody, includingthe CDR encoding sequences, were cloned and sequenced as described. Thenucleic acid and protein sequences of 3G8 are provided below. Using themouse variable region and CDR sequences, a large number of chimeric andhumanized monoclonal antibodies, comprising complementary determiningregions derived from 3G8 CDRs were produced and their propertiesanalyzed. To identify humanized antibodies that bind CD16A with highaffinity and have other desirable properties, antibody heavy chainscomprising a VH region with CDRs derived from 3G8 were produced andcombined (by coexpression) with antibody light chains comprising a VLregion with CDRs derived from 3G8 to produce a tetrameric antibody foranalysis. Properties of the resulting tetrameric antibodies weredetermined as described below. As described below, CD16A bindingproteins comprising 3G8 CDRs, such as the humanized antibody proteinsdescribed herein, may be used according to the invention.

5.1.2.1.1 VH Region

In one aspect, the CD16A binding protein of the invention may comprise aheavy chain variable domain in which at least one CDR (and usually threeCDRS) have the sequence of a CDR (and more typically all three CDRS) ofthe mouse monoclonal antibody 3G8 heavy chain and for which theremaining portions of the binding protein are substantially human(derived from and substantially similar to, the heavy chain variableregion of a human antibody or antibodies).

In an aspect, the invention provides a humanized 3G8 antibody orantibody fragment containing CDRs derived from the 3G8 antibody in asubstantially human framework, but in which at least one of the CDRs ofthe heavy chain variable domain differs in sequence from thecorresponding mouse antibody 3G8 heavy chain CDR. For example, in oneembodiment, the CDR(S) differs from the 3G8 CDR sequence at least byhaving one or more CDR substitutions shown known in the art to affectbinding of 3G8 to CD16A, as known in the art or as disclosed in Tables 3and 4A-H. Suitable CD16 binding proteins may comprise 0, 1, 2, 3, or 4,or more of these substitutions (and often have from 1 to 4 of thesesubstitutions) and optionally can have additional substitutions as well.

TABLE 3 V_(H) Domain Substitutions Kabat No. Position RegionSubstitutions 1 2 FR1 Ile 2 5 FR1 Lys 3 10 FR1 Thr 4 30 FR1 Arg 5 34CDR1 Val 6 50 CDR2 Leu 7 52 CDR2 Phe or Tyr or Asp 8 54 CDR2 Asn 9 60CDR2 Ser 10 62 CDR2 Ser 11 70 FR3 Thr 12 94 FR3 Gln or Lys or Ala or His13 99 CDR3 Tyr 14 101 CDR3 Asp

TABLE 4A V_(H) Sequences Derived from 3G8 V_(H) * FR1 CDR1 FR2 CDR2 FR3CDR3 FR4 3G8VH A A A A A A A Ch3G8VH A A A A A A B HxC B A B A A A B CxHA A A A B A B Hu3G8VH-1 B A B A B A B Hu3G8VH-2 C A B A B A B Hu3G8VH-3D A B A B A B Hu3G8VH-4 B A B A C B B Hu3G8VH-5 B A B A C A B Hu3G8VH-6B B B A B B B Hu3G8VH-7 B B B A B A B Hu3G8VH-8 B A B A B C B Hu3G8VH-9B A B B B B B Hu3G8VH-10 B A B A B B B Hu3G8VH-11 B A B B B A BHu3G8VH-12 B A B C B A B Hu3G8VH-13 B A B D B A B Hu3G8VH-14 B A B E B AB Hu3G8VH-15 B A B A D A B Hu3G8VH-16 B A B A E A B Hu3G8VH-17 B A B A FA B Hu3G8VH-18 B A B A G A B Hu3G8VH-19 B A B A C C B Hu3G8VH-20 B B B CB A B Hu3G8VH-21 B A B A D B B Hu3G8VH-22 B B B C B C B Hu3G8VH-23 B B BC E C B Hu3G8VH-24 B B B C F C B Hu3G8VH-25 B B B C G C B Hu3G8VH-26 B BB C C C B Hu3G8VH-27 B B B C E D B Hu3G8VH-28 B B B C F D B Hu3G8VH-29 BB B C G D B Hu3G8VH-30 B B B C C D B Hu3G8VH-31 E B B C B A B Hu3G8VH-32E B B H B A B Hu3G8VH-33 E B B H B A B Hu3G8VH-34 E B B C B C BHu3G8VH-35 E B B C C C B Hu3G8VH-36 E B B H C D B Hu3G8VH-37 E B B H E CB Hu3G8VH-38 E B B F B A B Hu3G8VH-39 E B B I B A B Hu3G8VH-40 E B B G BA B Hu3G8VH-41 E B B J B A B Hu3G8VH-42 E B B C H A B Hu3G8VH-43 E B B CH C B Hu3G8VH-44 E B B C I D B Hu3G8VH-45 E B B C J D B * Letters inTable 4A refer to sequences in Tables 4 B-H.

TABLE 4B FR1 A B C D E RESIDUE Q Q Q Q Q 1 V V V V I 2 T T T T T 3 L L LL L 4 K R K R K 5 E E E E E 6 S S S S S 7 G G G G G 8 P P P P P 9 G A AA T 10 I L L L L 11 L V V V V 12 Q K K K K 13 P P P P P 14 S T T T T 15Q Q Q Q Q 16 T T T T T 17 L L L L L 18 S T T T T 19 L L L L L 20 T T T TT 21 C C C C C 22 S T T T T 23 F F F F F 24 S S S S S 25 G G G G G 26 FF F F F 27 S S S S S 28 L L L L L 29 R S S R S 30 103 104 105 106 107SEQ ID NO. SEQ ID NO. Sequence 103 QVTLKESGPGILQPSQTLSLTCSFSGFSLR 104QVTLRESGPALVKPTQTLTLTCTFSGFSLS 105 QVTLKESGPALVKPTQTLTLTCTFSGFSLS 106QVTLRESGPALVKPTQTLTLTCTFSGFSLR 107 QITLKESGPTLVKPTQTLTLTCTFSGFSLS

TABLE 4C CDR1 A B RESIDUE T T 31 S S 32 G G 33 M V 34 G G 35 V V  35A GG  35B 108 109 SEQ ID NO. SEQ ID NO. Sequence 108 TSGMGVG 109 TSGVGVG

TABLE 4D FR2 A B RESIDUE W W 36 I I 37 R R 38 Q Q 39 P P 40 S P 41 G G42 K K 43 G A 44 L L 45 E E 46 W W 47 L L 48 A A 49 110 111 SEQ ID NO.SEQ ID NO. Sequence 110 WIRQPSGKGLEWLA 111 WIRQPPGKALEWLA

TABLE 4E CDR2 A B C D E F G H I J RESIDUE H H H H H L H L H L 50 I I I II I I I I I 51 W Y W Y W D F W D W 52 W W W W W W W W W W 53 D N D D N DD D D N 54 D D D D D D D D D D 55 D D D D D D D D D D 56 K K K K K K K KK K 57 R R R R R R R R R R 58 Y Y Y Y Y Y Y Y Y Y 59 N N S N N S S S S S60 P P P P P P P P P P 61 A A S A A S S S S 5 62 L L L L L L L L L L 63K K K K K K K K K K 64 S S S S S S S S S 5 65 112 113 114 115 116 117118 119 120 121 SEQ ID NO SEQ ID NO. Sequence 112 HIWWDDDKRYNPALKS 113HIYWNDDKRYNPALKS 114 HIWWDDDKRYSPSLKS 115 HIYWDDDKRYNPALKS 116HIWWNDDKRYNPALKS 117 LIDWDDDKRYSPSLKS 118 HIFWDDDKRYSPSLKS 119LIWWDDDKRYSPSLKS 120 HIDWDDDKRYSPSLKS 121 LIWWNDDKRYSPSLKS

TABLE 4F FR3 A B C D E F G H I J RESIDUE R R R R R R R R R R 66 L L L LL L L L L L 67 T T T T T T T T T T 68 I I I I I I I I I I 69 S S S S S SS T T T 70 K K K K K K K K K K 71 D D D D D D D D D D 72 T T T T T T T TT T 73 S S S S S S S S S S 74 S K K K K K K K K K 75 N N N N N N N N N N76 Q Q Q Q Q Q Q Q Q Q 77 V V V V V V V V V V 78 F V V V V V V V V V 79L L L L L L L L L L 80 K T T T T T T T T T 81 I M M M M M M M M M 82 A TT T T T T T T T  82A S N N N N N N N N N  82B V M M M M M M M M M  82C DD D D D D D D D D 83 T P P P P P P P P P 84 A V V V V V V V V V 85 D D DD D D D D D D 86 T T T T T T T T T T 87 A A A A A A A A A A 88 T T T T TT T T T T 89 Y Y Y Y Y Y Y Y Y Y 90 Y Y Y Y Y Y Y Y Y Y 91 C C C C C C CC C C 92 A A A A A A A A A A 93 Q R Q T K A H R H Q 94 122 123 124 125126 127 128 129 130 131 82 SEQ ID NO. 132 133 134 135 136 137 138 139140 141 82A SEQ ID NO. 142 143 144 145 146 147 148 149 150 151 82B SEQID NO. 152 153 154 155 156 157 158 159 160 161 82C SEQ ID NO. SEQ ID NO.Sequence 122 RLTISKDTSSNQVFLKIDTADTATYYCAQ 123RLTISKDTSKNQVVLTMDPVDTATYYCAR 124 RLTISKDTSKNQVVLTMDPVDTATYYCAQ 125RLTISKDTSKNQVVLTMDPVDTATYYCAT 126 RLTISKDTSKNQVVLTMDPVDTATYYCAK 127RLTISKDTSKNQVVLTMDPVDTATYYCAA 128 RLTISKDTSKNQVVLTMDPVDTATYYCAH 129RLTITKDTSKNQVVLTMDPVDTATYYCAR 130 RLTITKDTSKNQVVLTMDPVDTATYYCAH 131RLTITKDTSKNQVVLTMDPVDTATYYCAQ 132 RLTISKDTSSNQVFLKADTADTATYYCAQ 133RLTISKDTSKNQVVLTTDPVDTATYYCAR 134 RLTISKDTSKNQVVLTTDPVDTATYYCAQ 135RLTISKDTSKNQVVLTTDPVDTATYYCAT 136 RLTISKDTSKNQVVLTTDPVDTATYYCAK 137RLTISKDTSKNQVVLTTDPVDTATYYCAA 138 RLTISKDTSKNQVVLTTDPVDTATYYCAH 139RLTITKDTSKNQVVLTTDPVDTATYYCAR 140 RLTITKDTSKNQVVLTTDPVDTATYYCAH 141RLTITKDTSKNQVVLTTDPVDTATYYCAQ 142 RLTISKDTSSNQVFLKSDTADTATYYCAQ 143RLTISKDTSKNQVVLTNDPVDTATYYCAR 144 RLTISKDTSKNQVVLTNDPVDTATYYCAQ 145RLTISKDTSKNQVVLTNDPVDTATYYCAT 146 RLTISKDTSKNQVVLTNDPVDTATYYCAK 147RLTISKDTSKNQVVLTNDPVDTATYYCAA 148 RLTISKDTSKNQVVLTNDPVDTATYYCAH 149RLTITKDTSKNQVVLTNDPVDTATYYCAR 150 RLTITKDTSKNQVVLTNDPVDTATYYCAH 151RLTITKDTSKNQVVLTNDPVDTATYYCAQ 152 RLTISKDTSSNQVFLKVDTADTATYYCAQ 153RLTISKDTSKNQVVLTMDPVDTATYYCAR 154 RLTISKDTSKNQVVLTMDPVDTATYYCAQ 155RLTISKDTSKNQVVLTMDPVDTATYYCAT 156 RLTISKDTSKNQVVLTMDPVDTATYYCAK 157RLTISKDTSKNQVVLTMDPVDTATYYCAA 158 RLTISKDTSKNQVVLTMDPVDTATYYCAH 159RLTITKDTSKNQVVLTMDPVDTATYYCAR 160 RLTITKDTSKNQVVLTMDPVDTATYYCAH 161RLTITKDTSKNQVVLTMDPVDTATYYCAQ

TABLE 4G CDR3 A B C D RESIDUE I I I I 95 N N N N 96 P P P P 97 A A A A98 W W Y Y 99 F F F F 100 A D A D 101 Y Y Y Y 102 162 163 164 165 SEQ IDNO SEQ ID NO. Sequence 162 INPAWFAY 163 INPAWFDY 164 INPAYFAY 165INPAYFDY

TABLE 4H FR4 A B RESIDUE W W 103 G G 104 Q Q 105 G G 106 T T 107 L L 108V V 109 T T 110 V V 111 S S 112 A S 113 166 167 SEQ ID NO SEQ ID NO.Sequence 166 WGQGTLVTVSA 167 WGQGTLVTVSS

In one embodiment, a CD16A binding protein may comprise a heavy chainvariable domain sequence that is the same as, or similar to, the VHdomain of the Hu3G8VH-1 construct, the sequence of which is provided inSEQ ID NO: 68. For example, the invention provides a CD16A bindingprotein comprising a VH domain with a sequence that (1) differs from theVH domain of Hu3G8VH-1 (SEQ ID NO: 68) by zero, one, or more than one ofthe CDR substitutions set forth in Table 1; (2) differs from the VHdomain of Hu3G8VH-1 by zero, one or more than one of the frameworksubstitutions set forth in Table 1; and (3) is at least about 80%identical, often at least about 90%, and sometimes at least about 95%identical, or even at least about 98% identical to the Hu3G8VH-1 VHsequence at the remaining positions.

Exemplary VH domains of CD16 binding proteins of the invention have thesequence of 3G8VH, Hu3G8VH-5 and Hu3G8VH-22 (SEQ ID NO: 79, SEQ ID NO:69 and SEQ ID NO: 70, respectively). Exemplary nucleotide sequencesencoding the sequences of 3G8VH and Hu3G8VH-5 (SEQ ID NO: 79 and SEQ IDNO: 69, respectively) are provided by SEQ ID NO: 80 and SEQ ID NO: 81,respectively.

The VH domain may have a sequence that differs from that of Hu3G8VH-1(SEQ ID NO: 68) by at least one, at least two, at least three, at leastfour 4, at least five, or at least six of the substitutions shown inTable 3. These substitutions are believed to result in increasedaffinity for CD16A and/or reduce the immunogenicity of a CD16A bindingprotein when administered to humans. In certain embodiments, the degreeof sequence identity with the Hu3G8VH-1 VH domain at the remainingpositions is at least about 80%, at least about 90%, at least about 95%or at least about 98%.

For illustration and not limitation, the sequences of a number of CD16Abuilding protein VH domains is shown in Table 4. Heavy chains comprisingthese sequences fused to a human Cγ1 constant region were coexpressedwith the hu3G8VL-1 light chain (described below) to form tetramericantibodies, and binding of the antibodies to CD16A was measured toassess the effect of amino acid substitutions compared to the hu3G8VH-1VH domain. Constructs in which the VH domain has a sequence ofhu3G8VH-1, 2, 3, 4, 5, 8, 12, 14, 16, 17, 18, 19, 20, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 42, 43, 44 and 45 showedhigh affinity binding, with hu3G8VH-6 and -40 VH domains showingintermediate binding. CD16A binding proteins comprising the VH domainsof hu3G8VH-5 and hu3G8VH-22 (SEQ ID NO: 69 and SEQ ID NO: 70,respectively) are considered to have particularly favorable bindingproperties.

5.1.2.2 VL Region

Similar studies were conducted to identify light chain variable domainsequences with favorable binding properties. In one aspect, theinvention provides a CD16A binding protein containing a light chainvariable domain in which at least one CDR (and usually three CDRs) hasthe sequence of a CDR (and more typically all three CDRs) of the mousemonoclonal antibody 3G8 light chain and for which the remaining portionsof the binding protein are substantially human (derived from andsubstantially similar to, the heavy chain variable region of a humanantibody or antibodies).

In one aspect, the invention provides a fragment of a humanized 3G8antibody containing CDRs derived from the 3G8 antibody in asubstantially human framework, but in which at least one of the CDRs ofthe light chain variable domain differs in sequence from the mousemonoclonal antibody 3G8 light chain CDR. In one embodiment, the CDR(s)differs from the 3G8 sequence at least by having one or more amino acidsubstitutions in a CDR, such as, one or more substitutions shown inTable 2 (e.g., arginine at position 24 in CDR1; serine at position 25 inCDR1; tyrosine at position 32 in CDR1; leucine at position 33 in CDR1;aspartic acid, tryptophan or serine at position 50 in CDR2; serine atposition 53 in CDR2; alanine or glutamine at position 55 in CDR2;threonine at position 56 in CDR2; serine at position 93 in CDR3; and/orthreonine at position 94 in CDR3). In various embodiments, the variabledomain can have 0, 1, 2, 3, 4, 5, or more of these substitutions (andoften have from 1 to 4 of these substitutions) and optionally, can haveadditional substitutions as well.

In one embodiment, a suitable CD16A binding protein may comprise a lightchain variable domain sequence that is the same as, or similar to, theVL domain of the Hu3G8VL-1 (SEQ ID NO: 71) construct, the sequence ofwhich is provided in Table 6. For example, the invention provides aCD16A binding protein comprising a VL domain with a sequence that (1)differs from the VL domain of Hu3G8VL-1 (SEQ ID NO: 71) by zero, one, ormore of the CDR substitutions set forth in Table 5; (2) differs from theVL domain of Hu3G8VL-1 by zero, one or more of the frameworksubstitutions set forth in Table 5; and (3) is at least about 80%identical, often at least about 90%, and sometimes at least about 95%identical, or even at least about 98% identical to the Hu3G8VL-1 VLsequence (SEQ ID NO: 71) at the remaining positions.

TABLE 5 3G8 V_(L) Domain Substitutions Kabat No. Position RegionSubstitutions 1 24 CDR1 Arg 2 25 CDR1 Ser 3 32 CDR1 Tyr 4 33 CDR1 Leu 550 CDR2 Asp or Trp or Ser 6 51 CDR2 Ala 7 53 CDR2 Ser 8 55 CDR2 Ala orGln 9 56 CDR2 Thr 10 93 CDR3 Ser 11 94 CDR3 Thr

TABLE 6 V_(L) Sequences Derived from 3G8 V_(L) * FR1 CDR1 FR2 CDR2 FR3CDR3 FR4 3G8VL A A A A A A A Ch3G8VL A A A A A A A Hu3G8VL-1 B A A A B AB Hu3G8VL-2 B B A A B A B Hu3G8VL-3 B C A A B A B Hu3G8VL-4 B D A A B AB Hu3G8VL-5 B E A A B A B Hu3G8VL-6 B F A A B A B Hu3G8VL-7 B G A A B AB Hu3G8VL-8 B A A B B A B Hu3G8VL-9 B A A C B A B Hu3G8VL-10 B A A D B AB Hu3G8VL-11 B A A E B A B Hu3G8VL-12 B A A F B A B Hu3G8VL-13 B A A G BA B Hu3G8VL-14 B A A A B B B Hu3G8VL-15 B A A A B C B Hu3G8VL-16 B A A AB D B Hu3G8VL-17 B A A A B E B Hu3G8VL-18 B B A D B A B Hu3G8VL-19 B B AD B D B Hu3G8VL-20 B B A D B E B Hu3G8VL-21 B C A D B A B Hu3G8VL-22 B CA D B D B Hu3G8VL-23 B C A D B E B Hu3G8VL-24 B D A D B A B Hu3G8VL-25 BD A D B D B Hu3G8VL-26 B D A D B E B Hu3G8VL-27 B E A D B A B Hu3G8VL-28B E A D B D B Hu3G8VL-29 B E A D B E B Hu3G8VL-30 B A A D B D BHu3G8VL-31 B A A D B E B Hu3G8VL-32 B A A H B A B Hu3G8VL-33 B A A I B AB Hu3G8VL-34 B A A J B A B Hu3G8VL-35 B B A H B D B Hu3G8VL-36 B C A H BD B Hu3G8VL-37 B E A H B D B Hu3G8VL-38 B B A I B D B Hu3G8VL-39 B C A IB D B Hu3G8VL-40 B E A I B D B Hu3G8VL-41 B B A J B D B Hu3G8VL-42 B C AJ B D B Hu3G8VL-43 B E A J B D B Hu3G8VL-44 B A A K B A B *Letters inTable 6A refer to sequences in Tables 6B-H.

TABLE 6B FR1 A B RESIDUE D D 1 T I 2 V V 3 L M 4 T T 5 Q Q 6 S S 7 P P 8A D 9 S S 10 L L 11 A A 12 V V 13 S S 14 L L 15 G G 16 Q E 17 R R 18 A A19 T T 20 I I 21 S N 22 C C 23 168 169 SEQ ID NO SEQ ID NO. Sequence 168DTVLTQSPASLAVSL 169 DIVMTQSPDSLAVSL

TABLE 6C CDR1 A B C D E F G RESIDUE K R K K K K K 24 A A S A A A A 25 SS S S S S S 26 Q Q Q Q Q Q Q 27 S S S S S S S  27A V V V V V V V  27B DD D D D D D  27C F F F F F F F  27D D D D D D D D 28 G G G G G G G 29 DD D D D D D 30 S S S S S S S 31 F F F Y F F Y 32 M M M M L M L 33 N N NN N A A 34 170 171 172 173 174 175 176 27 SEQ ID NO 177 178 179 180 181182 183 27A SEQ ID NO 184 185 186 187 188 189 190 27B SEQ ID NO 191 192193 194 195 196 197 27C SEQ ID NO 198 199 200 201 202 203 204 27D SEQ IDNO SEQ ID NO. Sequence 170 KASQDGDSFMN 171 RASQDGDSFMN 172 KSSQDGDSFMN173 KASQDGDSYMN 174 KASQDGDSFLN 175 KASQDGDSFMA 176 KASQDGDSYLA 177KASSDGDSFMN 178 RASSDGDSFMN 179 KSSSDGDSFMN 180 KASSDGDSYMN 181KASSDGDSFLN 182 KASSDGDSFMA 183 KASSDGDSYLA 184 KASVDGDSFMN 185RASVDGDSFMN 186 KSSVDGDSFMN 187 KASVDGDSYMN 188 KASVDGDSFLN 189KASVDGDSFMA 190 KASVDGDSYLA 191 KASDDGDSFMN 192 RASDDGDSFMN 193KSSDDGDSFMN 194 KASDDGDSYMN 195 KASDDGDSFLN 196 KASDDGDSFMA 197KASDDGDSYLA 198 KASFDGDSFMN 199 RASFDGDSFMN 200 KSSFDGDSFMN 201KASFDGDSYMN 202 KASFDGDSFLN 203 KASFDGDSFMA 204 KASFDGDSYLA

TABLE 6D FR2 A RESIDUE W 35 Y 36 Q 37 Q 38 K 39 P 40 G 41 Q 42 P 43 P 44K 45 L 46 L 47 I 48 Y 49 205 SEQ ID NO SEQ ID NO. Sequence 205WYQQKAPGQPPKLLIY

TABLE 6E CDR2 A B C D E F G H I J K RESIDUE T D W T D D S S S T T 50 T AA T A A A T T T T 51 S S S S S S S S S S S 52 N N N N N N N N N N S 53 LL L L L L L L L L L 54 E E E E E A Q E Q Q Q 55 S S S T T T S S S S S 56206 207 208 209 210 211 212 213 214 215 216 SEQ ID NO SEQ ID NO.Sequence 206 TTSNLES 207 DASNLES 208 WASNLES 209 TTSNLET 210 DASNLET 211DASNLAT 212 SASNLQS 213 STSNLES 214 STSNLQS 215 TTSNLQS 216 TTSSLQS

TABLE 6F FR3 A B RESIDUE G G 57 I V 58 P P 59 A D 60 R R 61 F F 62 S S63 A G 64 S S 65 G G 66 S S 67 G G 68 T T 69 D D 70 F F 71 T T 72 L L 73N T 74 I I 75 H S 76 P S 77 V L 78 E Q 79 E A 80 E E 81 D D 82 T V 83 AA 84 T V 85 Y Y 86 Y Y 87 C C 88 217 218 SEQ ID NO SEQ ID NO. Sequence217 GIPARFSASGSGTDFTLNIHPVEEEDTATYYC 218GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

TABLE 6G CDR3 A B C D E RESIDUE Q Q Q Q Q 89 Q Q Q Q Q 90 S S S S S 91 NY Y N N 92 E S E S E 93 D T D D T 94 P P P P P 95 Y Y Y Y Y 96 T T T T T97 219 220 221 222 223 SEQ ID NO SEQ ID NO. Sequence 219 QQSNEDPYT 220QQSYSTPYT 221 QQSYEDPYT 222 QQSNSDPYT 223 QQSNETPYT

TABLE 6H FR4 A B RESIDUE F F 98 G G 99 G Q 100 G G 101 T T 102 K K 103 LL 104 E E 105 I I 106 K K 107 224 225 SEQ ID NO SEQ ID NO. Sequence 224FGGGTKLEIK 225 FGQGTKLEIK

Exemplary VL domains of CD16 binding proteins of the invention have thesequence of 3G8VL, Hu3G8VL-1 or Hu3G8VL-43, (SEQ ID NO: 82, SEQ ID NO:71 and SEQ ID NO: 72, respectively) as shown in Tables 5 and 6.Exemplary nucleotide sequences encoding 3G8VL (SEQ ID NO: 82) andHu3G8VL-1 (SEQ ID NO: 71) are provided in SEQ ID NO: 83 and SEQ ID NO:84, respectively.

The VL domain may have a sequence that differs from that of Hu3G8VL-1(SEQ ID NO: 71) by zero, one, at least two, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, or at least 9 of thesubstitutions shown in Table 2. These substitutions are believed toresult in increased affinity for CD16A and/or reduce the immunogenicityof a CD16A binding protein when administered to humans. In certainembodiments, the degree of sequence identity at the remaining positionsis at least about 80%, at least about 90% at least about 95% or at leastabout 98%.

For illustration and not limitation, the sequences of a number of CD16Abinding proteins VL domains is shown in Table 6. Light chains comprisingthese sequences fused to a human Cκ constant domain were coexpressedwith a Hu3G8VH heavy chain (described above) to form tetramericantibodies, and the binding of the antibodies to CD16A was measured toassess the effect of amino acid substitutions compared to the Hu3G8VL-1VL domain (SEQ ID NO: 71). Constructs in which the VL domain has asequence of hu3G8VL-1, 2, 3, 4, 5, 10, 16, 18, 19, 21, 22, 24, 27, 28,32, 33, 34, 35, 36, 37, and 42 showed high affinity binding andhu3G8VL-15, 17, 20, 23, 25, 26, 29, 30, 31, 38, 39, 40 and 41 showedintermediate binding. CD16A binding proteins comprising the VL domainsof hu3G8VL-1, hu3G8VL-22, and hu3G8VL-43 are considered to haveparticularly favorable binding properties (SEQ ID NO: 71, SEQ ID NO: 73and SEQ ID NO: 72, respectively).

5.1.2.2.1 Combinations of VL and/or VH Domains

As is known in the art and described elsewhere herein, immunoglobulinlight and heavy chains can be recombinantly expressed under conditionsin which they associate to produce a diabody, or can be so combined invitro. It will thus be appreciated that a 3G8-derived VL-domaindescribed herein can be combined a 3G8-derived VH-domain describedherein to produce a CD16A binding diabody, and all such combinations arecontemplated.

For illustration and not for limitation, examples of useful CD16Adiabodies are those comprising at least one VH domain and at least oneVL domain, where the VH domain is from hu3G8VH-1, hu3G8VH-22 orhu3G8VH-5 (SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 69, respectively)and the VL domain is from hu3G8VL-1, hu3G8VL-22 or hu3G8VL-43 (SEQ IDNO: 71, SEQ ID NO: 73 and SEQ ID NO: 41, respectively). In particular,humanized antibodies that comprise hu3G8VH-22 (SEQ ID NO: 22) andeither, hu3G8VL-1, hu3G8VL-22 or hu3G8VL-43 (SEQ ID NO: 71, SEQ ID NO:70 and SEQ ID NO: 72, respectively), or hu3G8VH-5 (SEQ ID NO: 69) andhu3G8VL-1 (SEQ ID NO: 71) have favorable properties.

It will be appreciated by those of skill that the sequences of VL and VHdomains described here can be further modified by art-known methods suchas affinity maturation (see Schier et al. (1996) “Isolation Of PicomolarAffinity Anti-C-ErbB-2 Single-Chain Fv By Molecular Evolution Of TheComplementarity Determining Regions In The Center Of The AntibodyBinding Site,” J. Mol. Biol. 263:551-567; Daugherty et al. (1998)“Antibody Affinity Maturation Using Bacterial Surface Display,” ProteinEng. 11:825-832; Boder et al. (1997) “Yeast Surface Display ForScreening Combinatorial Polypeptide Libraries,” Nat. Biotechnol.15:553-557; Boder et al. (2000) “Directed Evolution Of AntibodyFragments With Monovalent Femtomolar Antigen-Binding Affinity,” Proc.Natl. Acad. Sci. U.S.A 97:10701-10705; Hudson et al. (2003) “EngineeredAntibodies,” Nature Medicine 9:129-39). For example, the CD16A bindingproteins can be modified using affinity maturation techniques toidentify proteins with increased affinity for CD16A and/or decreasedaffinity for CD16B.

One exemplary CD16 binding protein is the mouse 3G8 antibody. Amino acidsequence comprising the VH and VL domains of humanized 3G8 are describedin FIGS. 2, 9, 14 and set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

5.2 Diabodies Comprising Fc Regions or Portions Thereof

The invention encompasses diabody molecules comprising Fc domains orportions thereof (e.g., a CH2 or CH3 domain). In certain embodiments,the Fc domain, or portion(s) thereof, comprises one or more constantdomain(s) of the Fc region of IgG2, IgG3 or IgG4 (e.g., CH2 or CH3). Inother embodiments, the invention encompasses molecules comprising and Fcdomain or portion thereof, wherein said Fc domain or portion thereofcomprises at least one amino acid modification (e.g. substitution)relative to a comparable wild-type Fc domain or portion thereof. VariantFc domains are well known in the art, and are primarily used to alterthe phenotype of the antibody comprising said variant Fc domain asassayed in any of the binding activity or effector function assays wellknown in the art, e.g. ELISA, SPR analysis, or ADCC. Such variant Fcdomains, or portions thereof, have use in the present invention byconferring or modifying the effector function exhibited by a diabodymolecule of the invention comprising an Fc domain (or portion thereof)as functionally assayed, e.g., in an NK dependent or macrophagedependent assay. Fc domain variants identified as altering effectorfunction are disclosed in International Application WO04/063351, U.S.Patent Application Publications 2005/0037000 and 2005/0064514, U.S.Provisional Applications 60/626,510, filed Nov. 10, 2004, 60/636,663,filed Dec. 15, 2004, and 60/781,564, filed Mar. 10, 2006, and U.S.patent application Ser. Nos. 11/271,140, filed Nov. 10, 2005, and11/305,787, filed Dec. 15, 2005, concurrent applications of theInventors, each of which is incorporated by reference in its entirety.

In other embodiments, the invention encompasses the use of any Fcvariant known in the art, such as those disclosed in Duncan et al.(1988) “Localization Of The Binding Site For The Human High-Affinity FcReceptor On IgG,” Nature 332:563-564; Lund et al. (1991) “Human Fc GammaRI And Fc Gamma RII Interact With Distinct But Overlapping Sites OnHuman IgG,” J. Immunol. 147:2657-2662; Lund et al. (1992) “MultipleBinding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma RII,” Mol.Immunol. 29:53-59; Alegre et al. (1994) “A Non-Activating “Humanized”Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties InVivo,” Transplantation 57:1537-1543; Hutchins et al. (1995) “ImprovedBiodistribution, Tumor Targeting, And Reduced Immunogenicity In MiceWith A Gamma 4 Variant Of Campath-1H” Proc. Natl. Acad. Sci. USA92:11980-11984; Jefferis et al. (1995) “Recognition Sites On Human IgGFor Fc Gamma Receptors: The Role Of Glycosylation,” Immunol. Lett.44:111-117; Lund et al. (1995) “Oligosaccharide-Protein Interactions InIgG Can Modulate Recognition By Fc Gamma Receptors,” FASEB J. 9:115-119;Jefferis et al. (1996) “Modulation Of Fc(Gamma)R And Human ComplementActivation By IgG3-Core Oligosaccharide Interactions,” Immunol. Lett.54:101-104; Lund et al. (1996) “Multiple Interactions Of Igg With ItsCore Oligosaccharide Can Modulate Recognition By Complement And Human FcGamma Receptor I And Influence The Synthesis Of Its OligosaccharideChains,” J. Immunol. 157:4963-4969; Armour et al. (1999) “RecombinantHuman IgG Molecules Lacking Fcgamma Receptor I Binding And MonocyteTriggering Activities,” Eur. J. Immunol. 29:2613-2624; Idusogie et al.(2000) “Mapping Of The C1Q Binding Site On Rituxan, A Chimeric AntibodyWith A Human IgG1 Fc,” J. Immunol. 164:4178-4184; Reddy et al. (2000)“Elimination Of Fc Receptor-Dependent Effector Functions Of A ModifiedIgG4 Monoclonal Antibody To Human CD4,” J. Immunol. 164:1925-1933; Xu etal. (2000) “In Vitro Characterization Of Five Humanized OKT3 EffectorFunction Variant Antibodies,” Cell. Immunol. 200:16-26; Idusogie et al.(2001) “Engineered Antibodies With Increased Activity To RecruitComplement,” J. Immunol. 166:2571-2575; Shields et al. (2001) “HighResolution Mapping Of The Binding Site On Human IgG1 For Fc gamma RI,Fc-gamma RII, Fc gamma RIII, And FcRn And Design Of IgG1 Variants WithImproved Binding To The Fc gamma R,” J. Biol. Chem. 276:6591-6604;Jefferis et al. (2002) “Interaction Sites On Human IgG-Fc For FcgammaR:Current Models,” Immunol. Lett. 82:57-65; Presta et al. (2002)“Engineering Therapeutic Antibodies For Improved Function,” Biochem.Soc. Trans. 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No.5,885,573; U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572;each of which is incorporated herein by reference in its entirety.

In certain embodiments, said one or more modifications to the aminoacids of the Fc region reduce the affinity and avidity of the Fc regionand, thus, the diabody molecule of the invention, for one or more FcγRreceptors. In a specific embodiment, the invention encompasses diabodiescomprising a variant Fc region, or portion thereof, wherein said variantFc region comprises at least one amino acid modification relative to awild type Fc region, which variant Fc region only binds one FcγR,wherein said FcγR is FcγRIIIA. In another specific embodiment, theinvention encompasses diabodies comprising a variant Fc region, orportion thereof, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region only binds one FcγR, wherein said FcγR is FcγRIIA. In anotherspecific embodiment, the invention encompasses diabodies comprising avariant Fc region, or portion thereof, wherein said variant Fc regioncomprises at least one amino acid modification relative to a wild typeFc region, which variant Fc region only binds one FcγR, wherein saidFcγR is FcγRIIB. In certain embodiments, the invention encompassesmolecules comprising a variant Fc domain wherein said variant confers ormediates increased ADCC activity and/or an increased binding to FcγRIIA(CD32A), relative to a molecule comprising no Fc domain or comprising awild-type Fc domain, as measured using methods known to one skilled inthe art and described herein. In alternate embodiments, the inventionencompasses molecules comprising a variant Fc domain wherein saidvariant confers or mediates decreased ADCC activity (or other effectorfunction) and/or an increased binding to FcγRIIB (CD32B), relative to amolecule comprising no Fc domain or comprising a wild-type Fc domain, asmeasured using methods known to one skilled in the art and describedherein.

The invention also encompasses the use of an Fc domain comprisingdomains or regions from two or more IgG isotypes. As known in the art,amino acid modification of the Fc region can profoundly affectFc-mediated effector function and/or binding activity. However, thesealterations in functional characteristics can be further refined and/ormanipulated when implemented in the context of selected IgG isotypes.Similarly, the native characteristics of the isotype Fc may bemanipulated by the one or more amino acid modifications. The multipleIgG isotypes (i.e., IgG1, IgG2, IgG3 and IgG4) exhibit differingphysical and functional properties including serum half-life, complementfixation, FcγR binding affinities and effector function activities (e.g.ADCC, CDC) due to differences in the amino acid sequences of their hingeand/or Fc domains. In certain embodiments, the amino acid modificationand IgG Fc region are independently selected based on their respective,separate binding and/or effector function activities in order toengineer a diabody with desired characteristics. In most embodiments,said amino acid modifications and IgG hinge/Fc regions have beenseparately assayed for binding and/or effector function activity asdescribed herein or known in the art in an the context of an IgG1. Incertain embodiments, said amino acid modification and IgG hinge/Fcregion display similar functionality, e.g., increased affinity forFcγRIIA, when separately assayed for FcγR binding or effector functionin the context of the diabody molecule or other Fc-containing molecule(e.g. and immunoglobulin). The combination of said amino acidmodification and selected IgG Fc region then act additively or, morepreferably, synergistically to modify said functionality in the diabodymolecule of the invention, relative to a diabody molecule of theinvention comprising a wild-type Fc region. In other embodiments, saidamino acid modification and IgG Fc region display oppositefunctionalities, e.g., increased and decreased, respectively, affinityfor FcγRIIA, when separately assayed for FcγR binding and/or effectorfunction in the context of the diabody molecule or other Fc containingmolecule (e.g., an immunoglobulin) comprising a wild-type Fc region asdescribed herein or known in the art; the combination of said “opposite”amino acid modification and selected IgG region then act to selectivelytemper or reduce a specific functionality in the diabody of theinvention relative to a diabody of the invention not comprising an Fcregion or comprising a wild-type Fc region of the same isotype.Alternatively, the invention encompasses variant Fc regions comprisingcombinations of amino acid modifications known in the art and selectedIgG regions that exhibit novel properties, which properties were notdetectable when said modifications and/or regions were independentlyassayed as described herein.

The functional characteristics of the multiple IgG isotypes, and domainsthereof, are well known in the art. The amino acid sequences of IgG1,IgG2, IgG3 and IgG4 are presented in FIGS. 1A-1B. Selection and/orcombinations of two or more domains from specific IgG isotypes for usein the methods of the invention may be based on any known parameter ofthe parent isotypes including affinity to FcγR (Table 7; Flesch et al.(2000) “Functions Of The Fc Receptors For Immunoglobulin G,” J. Clin.Lab. Anal. 14:141-156; Chappel et al. (1993) “Identification Of ASecondary Fc Gamma RI Binding Site Within A Genetically Engineered HumanIgG Antibody,” J. Biol. Chem. 33:25124-25131; Chappel et al. (1991)“Identification Of The Fc Gamma Receptor Class I Binding Site In HumanIgG Through The Use Of Recombinant IgG1/IgG2 Hybrid And Point-MutatedAntibodies,” Proc. Natl. Acad. Sci. USA 88:9036-9040, each of which ishereby incorporated by reference in its entirety). For example, use ofregions or domains from IgG isotypes that exhibit limited or no bindingto FcγRIIB, e.g., IgG2 or IgG4, may find particular use where a diabodyis desired to be engineered to maximize binding to an activatingreceptor and minimize binding to an inhibitory receptor. Similarly, useof Fc regions or domains from IgG isotypes known to preferentially bindC1q or FcγRIIIA, e.g., IgG3 (Brüggemann et al. (1987) “Comparison Of TheEffector Functions Of Human Immunoglobulins Using A Matched Set OfChimeric Antibodies,” J. Exp. Med. 166:1351-1361), may be combined withFc amino acid modifications of known in the art to enhance ADCC, toengineer a diabody molecule such that effector function activity, e.g.,complement activation or ADCC, is maximized.

TABLE 7 General characteristics of IgG binding to FcγR, adapted fromFlesch and Neppert, 1999, J. Clin. Lab. Anal. 14: 141-156 EstimatedAffinity for IgG Receptor (M⁻¹) Relative Affinity FcγRI 10⁸-10⁹ IgG3 >IgG1 >> IgG4 no-binding: IgG2 FcγRIIA R^(131 A) <10⁷ IgG3 > IgG1no-binding: IgG2, IgG4 FcγRIIA H^(131 A) <10⁷ IgG3 > IgG1 > IgG2no-binding: IgG4 FcγRIIB^(A) <10⁷ IgG3 > IgG1 > IgG4 no-binding: IgG2FcγRIII <10⁷ IgG3 = IgG1 no-binding: IgG2, IgG4 ^(A)binds only complexedIgG

5.3 Molecular Conjugates

The diabody molecules of the invention may be recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences.

Further, the diabody molecules of the invention (i.e., polypeptides) maybe conjugated to a therapeutic agent or a drug moiety that modifies agiven biological response. As an alternative to direct conjugation,owing to the multiple epitope binding sites on the multivalent, e.g.,tetravalent, diabody molecules of the invention, at least one bindingregion of the diabody may be designed to bind the therapeutic agent ordesired drug moiety without affecting diabody binding.

Therapeutic agents or drug moieties are not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin,gelonin, and pokeweed antiviral protein, a protein such as tumornecrosis factor, interferons including, but not limited to, α-interferon(IFN-α), β-interferon (IFN-β), nerve growth factor (NGF), plateletderived growth factor (PDGF), tissue plasminogen activator (TPA), anapoptotic agent (e.g., TNF-α, TNF-β, AIM I as disclosed in PCTPublication No. WO 97/33899), AIM II (see, PCT Publication No. WO97/34911), Fas ligand, and VEGI (PCT Publication No. WO 99/23105), athrombotic agent or an anti-angiogenic agent (e.g., angiostatin orendostatin), or a biological response modifier such as, for example, alymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”),macrophage colony stimulating factor, (“M-CSF”), or a growth factor(e.g., growth hormone (“GH”); proteases, or ribonucleases.

The diabody molecules of the invention (i.e., polypeptides) can be fusedto marker sequences, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al. (1989) “Bioassay For Trans-Activation Using Purified HumanImmunodeficiency Virus TAT-Encoded Protein: Trans-Activation RequiresmRNA Synthesis,” Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al. (1984)“The Structure Of An Antigenic Determinant In A Protein,” Cell,37:767-778) and the “flag” tag (Knappik et al. (1994) “An ImprovedAffinity Tag Based On The FLAG Peptide For The Detection AndPurification Of Recombinant Antibody Fragments,” Biotechniques,17(4):754-761).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of molecules of the invention (e.g.,epitope binding sites with higher affinities and lower dissociationrates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721;5,834,252; and 5,837,458, and Patten et al. (1997) “Applications Of DNAShuffling To Pharmaceuticals And Vaccines,” Curr. Opinion Biotechnol.8:724-733; Harayama (1998) “Artificial Evolution By DNA Shuffling,”Trends Biotechnol. 16:76-82; Hansson et al. (1999) “Evolution OfDifferential Substrate Specificities In Mu Class GlutathioneTransferases Probed By DNA Shuffling,” J. Mol. Biol. 287:265-276; andLorenzo et al. (1998) “PCR-Based Method For The Introduction OfMutations In Genes Cloned And Expressed In Vaccinia Virus,”BioTechniques 24:308-313 (each of these patents and publications arehereby incorporated by reference in its entirety). The diabody moleculesof the invention, or the nucleic acids encoding the molecules of theinvention, may be further altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. One or more portions of a polynucleotideencoding a molecule of the invention, may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

The present invention also encompasses diabody molecules of theinvention conjugated to or immunospecifically recognizing a diagnosticor therapeutic agent or any other molecule for which serum half-life isdesired to be increased/decreased and/or targeted to a particular subsetof cells. The molecules of the invention can be used diagnostically to,for example, monitor the development or progression of a disease,disorder or infection as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the molecules of the invention to a detectablesubstance or by the molecules immunospecifically recognizing thedetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to themolecules of the invention or indirectly, through an intermediate (suchas, for example, a linker known in the art) using techniques known inthe art, or the molecule may immunospecifically recognize the detectablesubstance: immunospecifically binding said substance. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Such diagnosis and detection can be accomplished designing the moleculesto immunospecifically recognize the detectable substance or by couplingthe molecules of the invention to detectable substances including, butnot limited to, various enzymes, enzymes including, but not limited to,horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin; fluorescent materialssuch as, but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent material such as, but not limitedto, luminol; bioluminescent materials such as, but not limited to,luciferase, luciferin, and aequorin; radioactive material such as, butnot limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),iodine (¹³¹In, ¹²⁵I, ¹²³I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹ Pm), rhenium (¹⁸⁶Re,¹⁸⁸Re), rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium(⁴⁷Sc), selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium(⁹⁹Tc), thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon(¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions.

The diabody molecules of the invention may immunospecifically recognizeor be conjugated to a therapeutic moiety such as a cytotoxin (e.g., acytostatic or cytocidal agent), a therapeutic agent or a radioactiveelement (e.g., alpha-emitters, gamma-emitters, etc.). Cytotoxins orcytotoxic agents include any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC), and anti-mitotic agents (e.g., vincristine andvinblastine).

Moreover, a diabody molecule of the invention can be conjugated to or bedesigned to immunospecifically recognize therapeutic moieties such as aradioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the polypeptide via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.(1998) “Comparison Of1,4,7,10-Tetraazacyclododecane-N,N′,N″,N′″-Tetraacetic Acid(DOTA)-Peptide-ChL6, A Novel Immunoconjugate With Catabolizable Linker,To 2-Iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 In BreastCancer Xenografts,” Clin. Cancer Res. 4:2483-2490; Peterson et al.(1999) “Enzymatic Cleavage Of Peptide-Linked Radiolabels FromImmunoconjugates,” Bioconjug. Chem. 10:553-; and Zimmerman et al, (1999)“A Triglycine Linker Improves Tumor Uptake And Biodistributions Of67-Cu-Labeled Anti-Neuroblastoma mAb chCE7 F(ab′)2 Fragments,” Nucl.Med. Biol. 26:943-950 each of which is incorporated herein by referencein their entireties.

Techniques for conjugating such therapeutic moieties to polypeptides,including e.g., Fc domains, are well known; see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), 1985, pp. 475-506); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,”Immunol. Rev., 62:119-158.

The diabody molecule of the invention may be administered with orwithout a therapeutic moiety conjugated to it, administered alone, or incombination with cytotoxic factor(s) and/or cytokine(s) for use as atherapeutic treatment. Where administered alone, at least one epitope ofa multivalent, e.g., tetravalent, diabody molecule may be designed toimmunospecifically recognize a therapeutic agent, e.g., cytotoxicfactor(s) and/or cytokine(s), which may be administered concurrently orsubsequent to the molecule of the invention. In this manner, the diabodymolecule may specifically target the therapeutic agent in a mannersimilar to direct conjugation. Alternatively, a molecule of theinvention can be conjugated to an antibody to form an antibodyheteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, whichis incorporated herein by reference in its entirety. Diabody moleculesof the invention may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.4 Characterization of Binding of Diabody Molecules

The diabody molecules of the present invention may be characterized in avariety of ways. In particular, molecules of the invention may beassayed for the ability to immunospecifically bind to an antigen, e.g.,FcRIIIA or FcRIIB, or, where the molecule comprises an Fc domain (orportion thereof) for the ability to exhibit Fc-FcγR interactions, i.e.specific binding of an Fc domain (or portion thereof) to an FcγR. Suchan assay may be performed in solution (e.g., Houghten (1992) “The Use OfSynthetic Peptide Combinatorial Libraries For The Identification OfBioactive Peptides,” BioTechniques, 13:412-421), on beads (Lam (1991) “ANew Type Of Synthetic Peptide Library For Identifying Ligand-BindingActivity,” Nature, 354:82-84, on chips (Fodor (1993) “MultiplexedBiochemical Assays With Biological Chips,” Nature, 364:555-556), onbacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), on plasmids (Cull et al. (1992) “ScreeningFor Receptor Ligands Using Large Libraries Of Peptides Linked To The CTerminus Of The Lac Repressor,” Proc. Natl. Acad. Sci. USA,89:1865-1869) or on phage (Scott et al. (1990) “Searching For PeptideLigands With An Epitope Library,” Science, 249:386-390; Devlin (1990)“Random Peptide Libraries: A Source Of Specific Protein BindingMolecules,” Science, 249:404-406; Cwirla et al. (1990) “Peptides OnPhage: A Vast Library Of Peptides For Identifying Ligands,” Proc. Natl.Acad. Sci. USA, 87:6378-6382; and Felici (1991) “Selection Of AntibodyLigands From A Large Library Of Oligopeptides Expressed On A MultivalentExposition Vector,” J. Mol. Biol., 222:301-310) (each of thesereferences is incorporated by reference herein in its entirety).Molecules that have been identified to immunospecifically bind to anantigen, e.g., FcγRIIIA, can then be assayed for their specificity andaffinity for the antigen.

Molecules of the invention that have been engineered to comprisemultiple epitope binding domains may be assayed for immunospecificbinding to one or more antigens (e.g., cancer antigen andcross-reactivity with other antigens (e.g., FcγR)) or, where themolecules comprise am Fc domain (or portion thereof) for Fc-FcγRinteractions by any method known in the art. Immunoassays which can beused to analyze immunospecific binding, cross-reactivity, and Fc-FcγRinteractions include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety).

The binding affinity and the off-rate of antigen-binding domaininteraction or Fc-FcγR interaction can be determined by competitivebinding assays. One example of a competitive binding assay is aradioimmunoassay comprising the incubation of labeled antigen, such astetrameric FcγR (e.g., ³H or ¹²⁵I, see Section 5.4.1) with a molecule ofinterest (e.g., molecules of the present invention comprising multipleepitope binding domains in the presence of increasing amounts ofunlabeled epitope, such as tetrameric FcγR (see Section 5.4.1), and thedetection of the molecule bound to the labeled antigen. The affinity ofthe molecule of the present invention for an antigen and the bindingoff-rates can be determined from the saturation data by Scatchardanalysis.

The affinities and binding properties of the molecules of the inventionfor an antigen or FcγR may be initially determined using in vitro assays(biochemical or immunological based assays) known in the art forantigen-binding domain or Fc-FcγR, interactions, including but notlimited to ELISA assay, surface plasmon resonance assay,immunoprecipitation assays. Preferably, the binding properties of themolecules of the invention are also characterized by in vitro functionalassays for determining one or more FcγR mediator effector cellfunctions, as described in section 5.4.2. In most preferred embodiments,the molecules of the invention have similar binding properties in invivo models (such as those described and disclosed herein) as those inin vitro based assays. However, the present invention does not excludemolecules of the invention that do not exhibit the desired phenotype inin vitro based assays but do exhibit the desired phenotype in vivo.

In some embodiments, screening and identifying molecules comprisingmultiple epitope binding domains and, optionally, Fc domains (orportions thereof) are done functional based assays, preferably in a highthroughput manner. The functional based assays can be any assay known inthe art for characterizing one or more FcγR mediated effector cellfunctions such as those described herein in Sections 5.4.2 and 5.4.3.Non-limiting examples of effector cell functions that can be used inaccordance with the methods of the invention, include but are notlimited to, antibody-dependent cell mediated cytotoxicity (ADCC),antibody-dependent phagocytosis, phagocytosis, opsonization,opsonophagocytosis, cell binding, rosetting, C1q binding, and complementdependent cell mediated cytotoxicity.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of molecules of the present invention to anantigen or and FcγR. BIAcore kinetic analysis comprises analyzing thebinding and dissociation of an antigen or FcγR from chips withimmobilized molecules (e.g., molecules comprising epitope bindingdomains or Fc domains (or portions thereof), respectively) on theirsurface. BIAcore analysis is described in Section 5.4.3.

Preferably, fluorescence activated cell sorting (FACS), using any of thetechniques known to those skilled in the art, is used for immunologicalor functional based assay to characterize molecules of the invention.Flow sorters are capable of rapidly examining a large number ofindividual cells that have been bound, e.g., opsonized, by molecules ofthe invention (e.g., 10-100 million cells per hour) (Shapiro et al.(1995) Practical Flow Cytometry). Additionally, specific parameters usedfor optimization of diabody behavior, include but are not limited to,antigen concentration (i.e., FcγR tetrameric complex, see Section5.4.1), kinetic competition time, or FACS stringency, each of which maybe varied in order to select for the diabody molecules comprisingmolecules of the invention which exhibit specific binding properties,e.g., concurrent binding to multiple epitopes. Flow cytometers forsorting and examining biological cells are well known in the art. Knownflow cytometers are described, for example, in U.S. Pat. Nos. 4,347,935;5,464,581; 5,483,469; 5,602,039; 5,643,796; and 6,211,477; the entirecontents of which are incorporated by reference herein. Other known flowcytometers are the FACS Vantage™ system manufactured by Becton Dickinsonand Company, and the COPAS™ system manufactured by Union Biometrica.

Characterization of target antigen binding affinity or Fc-FcγR bindingaffinity, and assessment of target antigen or FcγR density on a cellsurface may be made by methods well known in the art such as Scatchardanalysis or by the use of kits as per manufacturer's instructions, suchas Quantum™ Simply Cellular® (Bangs Laboratories, Inc., Fishers, Ind.).The one or more functional assays can be any assay known in the art forcharacterizing one or more FcγR mediated effector cell function as knownto one skilled in the art or described herein. In specific embodiments,the molecules of the invention comprising multiple epitope bindingdomains and, optionally, and Fc domain (or portion thereof) are assayedin an ELISA assay for binding to one or more target antigens or one ormore FcγRs, e.g., FcγRIIIA, FcγRIIA, FcγRIIA; followed by one or moreADCC assays. In some embodiments, the molecules of the invention areassayed further using a surface plasmon resonance-based assay, e.g.,BIAcore. Surface plasmon resonance-based assays are well known in theart, and are further discussed in Section 5.4.3, and exemplified herein,e.g., in Example 6.1.

In most preferred embodiments, the molecules of the invention comprisingmultiple epitope binding domains and, optionally, and Fc domain (orportion thereof) is further characterized in an animal model forinteraction with a target antigen (e.g., an FcγR) or for Fc-FcγRinteraction. Where Fc-FcγR interactions are to be assessed, preferredanimal models for use in the methods of the invention are, for example,transgenic mice expressing human FcγR5, e.g., any mouse model describedin U.S. Pat. Nos. 5,877,397, and 6,676,927 which are incorporated hereinby reference in their entirety. Further transgenic mice for use in suchmethods include, but are not limited to, nude knockout FcγRIIIA micecarrying human FcγRIIIA; nude knockout FcγRIIIA mice carrying humanFcγRIIA; nude knockout FcγRIIIA mice carrying human FcγRIIB and humanFcγRIIIA; nude knockout FcγRIIIA mice carrying human FcγRIIB and humanFcγRIIA; nude knockout FcγRIIIA and FcγRIIA mice carrying human FcγRIIIAand FcγRIIA and nude knockout FcγRIIIA, FcγRIIA and FcγRIIB micecarrying human FcγRIIIA, FcγRIIA and FcγRIIB.

5.4.1 Binding Assays Comprising FcγR

Characterization of binding to FcγR by molecules comprising an Fc domain(or portion thereof) and/or comprising epitope binding domain specificfor an FcγR may be done using any FcγR, including but not limited topolymorphic variants of FcγR. In some embodiments, a polymorphic variantof FcγRIIIA is used, which contains a phenylalanine at position 158. Inother embodiments, characterization is done using a polymorphic variantof FcγRIIIA which contains a valine at position 158. FcγRIIIA 158Vdisplays a higher affinity for IgG1 than 158F and an increased ADCCactivity (see, e.g., Koene et al. (1997) “Fc gammaRIIIa-158V/FPolymorphism Influences The Binding Of IgG By Natural Killer Cell FcgammaRIIIa, Independently Of The Fc gammaRIIIa-48L/R/H Phenotype,”Blood, 90:1109-14; Wu et al. (1997) “A Novel Polymorphism OfFcgammaRIIIa (CD16) Alters Receptor Function And Predisposes ToAutoimmune Disease,” J. Clin. Invest. 100: 1059-70, both of which areincorporated herein by reference in their entireties); this residue infact directly interacts with the lower hinge region of IgG1 as recentlyshown by IgG1-FcγRIIIA co-crystallization studies, see, e.g., Sondermannet al. (2000) “The 3.2-A Crystal Structure Of The Human IgG1 FcFragment-Fc gammaRIII complex,” Nature, 406(6793):267-273, which isincorporated herein by reference in its entirety. Studies have shownthat in some cases, therapeutic antibodies have improved efficacy inFcγRIIIA-158V homozygous patients. For example, humanized anti-CD20monoclonal antibody Rituximab was therapeutically more effective inFcγRIIIA 158V homozygous patients compared to FcγRIIIA 158F homozygouspatients (See, e.g., Cartron et al. (2002) “Therapeutic Activity OfHumanized Anti-CD20 Monoclonal Antibody And Polymorphism In IgG FcReceptor FcgammaRIIIA Gene,” Blood, 99(3): 754-758). In otherembodiments, therapeutic molecules comprising this region may also bemore effective on patients heterozygous for FcγRIIIA-158V andFcγRIIIA-158F, and in patients with FcγRIIA-131H. Although not intendingto be bound by a particular mechanism of action, selection of moleculesof the invention with alternate allotypes may provide for variants thatonce engineered into therapeutic diabodies will be clinically moreefficacious for patients homozygous for said allotype.

An FcγR binding assay was developed for determining the binding of themolecules of the invention to FcγR, and, in particular, for determiningbinding of Fc domains to FcγR. The assay allowed detection andquantitation of Fc-FcγR interactions, despite the inherently weakaffinity of the receptor for its ligand, e.g., in the micromolar rangefor FcγRIIB and FcγRIIIA. The method is described in detail inInternational Application WO04/063351 and U.S. Patent ApplicationPublications 2005/0037000 and 2005/0064514, each of which is herebyincorporated by reference in its entirety. Briefly, the method involvesthe formation of an FcγR complex that may lie sued in any standardimmunoassay known in the art, e.g., FACS, ELISA, surface plasmonresonance, etc. Additionally, the FcγR complex has an improved avidityfor an Fc region, relative to an uncomplexed FcγR. According to theinvention, the preferred molecular complex is a tetrameric immunecomplex, comprising: (a) the soluble region of FcγR (e.g., the solubleregion of FcγRIIIA, FcγRIIA or FcγRIIB); (b) a biotinylated 15 aminoacid AVITAG sequence (AVITAG) operably linked to the C-terminus of thesoluble region of FcγR (e.g., the soluble region of FcγRIIIA, FcγRIIA orFcγRIIB); and (c) streptavidin-phycoerythrin (SA-PE); in a molar ratioto form a tetrameric FcγR complex (preferably in a 5:1 molar ratio). Thefusion protein is biotinylated enzymatically, using for example, the E.coli Bir A enzyme, a biotin ligase which specifically biotinylates alysine residue in the 15 amino acid AVITAG sequence. The biotinylatedsoluble FcγR proteins are then mixed with SA-PE in a 1×SA-PE:5×biotinylated soluble FcγR molar ratio to form a tetrameric FcγR complex.

Polypeptides comprising Fc regions have been shown to bind thetetrameric FcγR complexes with at least an 8-fold higher affinity thanthe monomeric uncomplexed FcγR. The binding of polypeptides comprisingFc regions to the tetrameric FcγR complexes may be determined usingstandard techniques known to those skilled in the art, such as forexample, fluorescence activated cell sorting (FACS), radioimmunoassays,ELISA assays, etc.

The invention encompasses the use of the immune complexes comprisingmolecules of the invention, and formed according to the methodsdescribed above, for determining the functionality of moleculescomprising an Fc region in cell-based or cell-free assays.

As a matter of convenience, the reagents may be provided in an assaykit, i.e., a packaged combination of reagents for assaying the abilityof molecules comprising Fc regions to bind FcγR tetrameric complexes.Other forms of molecular complexes for use in determining Fc-FcγRinteractions are also contemplated for use in the methods of theinvention, e.g., fusion proteins-formed as described in U.S. ProvisionalApplication 60/439,709, filed on Jan. 13, 2003; which is incorporatedherein by reference in its entirety.

5.4.2 Functional Assays of Molecules with Variant Heavy Chains

The invention encompasses characterization of the molecules of theinvention comprising multiple epitope binding domains and, optionally,Fc domains (or portions thereof) using assays known to those skilled inthe art for identifying the effector cell function of the molecules. Inparticular, the invention encompasses characterizing the molecules ofthe invention for FcγR-mediated effector cell function. Additionally,where at least one of the target antigens of the diabody molecule of theinvention is an FcγR, binding of the FcγR by the diabody molecule mayserve to activate FcγR-mediated pathways similar to those activated byFcγR-Fc binding. Thus, where at least one eptiope binding domain of thediabody molecule recognizes an FcγR, the diabody molecule may elicitFcγR-mediated effector cell function without containing an Fc domain (orportion thereof), or without concomitant Fc-FcγR binding. Examples ofeffector cell functions that can be assayed in accordance with theinvention, include but are not limited to, antibody-dependent cellmediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis,C1q binding, and complement dependent cell mediated cytotoxicity. Anycell-based or cell free assay known to those skilled in the art fordetermining effector cell function activity can be used (For effectorcell assays, see Perussia et al. (2000) “Assays For Antibody-DependentCell-Mediated Cytotoxicity (ADCC) And Reverse ADCC (RedirectedCytotoxicity) In Human Natural Killer Cells,” Methods Mol. Biol. 121:179-92; Baggiolini et al. (1988) “Cellular Models For The Detection AndEvaluation Of Drugs That Modulate Human Phagocyte Activity,”Experientia, 44(10): 841-848; Lehmann et al. (2000) “Phagocytosis:Measurement By Flow Cytometry,” J. Immunol. Methods, 243(1-2): 229-42;Brown (1994) “In Vitro Assays Of Phagocytic Function Of Human PeripheralBlood Leukocytes: Receptor Modulation And Signal Transduction,” MethodsCell Biol., 45: 147-64; Munn et al. (1990) “Phagocytosis Of Tumor CellsBy Human Monocytes Cultured In Recombinant Macrophage Colony-StimulatingFactor,” J. Exp. Med., 172: 231-237, Abdul-Majid et al. (2002) “FcReceptors Are Critical For Autoimmune Inflammatory Damage To The CentralNervous System In Experimental Autoimmune Encephalomyelitis,” Scand. J.Immunol. 55: 70-81; Ding et al. (1998) “Two Human T Cell Receptors BindIn A Similar Diagonal Mode To The HLA-A2/Tax Peptide Complex UsingDifferent TCR Amino Acids,” Immunity 8:403-411, each of which isincorporated by reference herein in its entirety).

In one embodiment, the molecules of the invention can be assayed forFcγR-mediated phagocytosis in human monocytes. Alternatively, theFcγR-mediated phagocytosis of the molecules of the invention may beassayed in other phagocytes, e.g., neutrophils (polymorphonuclearleuckocytes; PMN); human peripheral blood monocytes, monocyte-derivedmacrophages, which can be obtained using standard procedures known tothose skilled in the art (e.g., see Brown (1994) “In Vitro Assays OfPhagocytic Function Of Human Peripheral Blood Leukocytes: ReceptorModulation And Signal Transduction,” Methods Cell Biol., 45: 147-164).In one embodiment, the function of the molecules of the invention ischaracterized by measuring the ability of THP-1 cells to phagocytosefluoresceinated IgG-opsonized sheep red blood cells (SRBC) by methodspreviously described (Tridandapani et al. (2000) “The Adapter ProteinLAT Enhances Fcgamma Receptor-Mediated Signal Transduction In MyeloidCells,” J. Biol. Chem. 275: 20480-20487).

Another exemplary assay for determining the phagocytosis of themolecules of the invention is an antibody-dependent opsonophagocytosisassay (ADCP) which can comprise the following: coating a targetbioparticle such as Escherichia coli-labeled FITC (Molecular Probes) orStaphylococcus aureus-FITC with (i) wild-type 4-4-20 antibody, anantibody to fluorescein (See Bedzyk et al. (1989) “Comparison OfVariable Region Primary Structures Within An Anti-Fluorescein IdiotypeFamily,” J. Biol. Chem., 264(3): 1565-1569, which is incorporated hereinby reference in its entirety), as the control antibody forFcγR-dependent ADCP; or (ii) 4-4-20 antibody harboring the D265Amutation that knocks out binding to FcγRIII, as a background control forFcγR-dependent ADCP (iii) a diabody comprising the epitope bindingdomain of 4-4-20 and an Fc domain and/or an epitope binding domainspecific for FcγRIII; and forming the opsonized particle; adding any ofthe opsonized particles described (i-iii) to THP-1 effector cells (amonocytic cell line available from ATCC) at a 1:1, 10:1, 30:1, 60:1,75:1 or a 100:1 ratio to allow FcγR-mediated phagocytosis to occur;preferably incubating the cells and E. coli-FITC/antibody at 37° C. for1.5 hour; adding trypan blue after incubation (preferably at roomtemperature for 2-3 min.) to the cells to quench the fluoroscence of thebacteria that are adhered to the outside of the cell surface withoutbeing internalized; transferring cells into a FACS buffer (e.g., 0.1%,BSA in PBS, 0.1%, sodium azide), analyzing the fluorescence of the THP1cells using FACS (e.g., BD FACS Calibur). Preferably, the THP-1 cellsused in the assay are analyzed by FACS for expression of FcγR on thecell surface. THP-1 cells express both CD32A and CD64. CD64 is a highaffinity FcγR that is blocked in conducting the ADCP assay in accordancewith the methods of the invention. The THP-1 cells are preferablyblocked with 100 μg/mL soluble IgG1 or 10% human serum. To analyze theextent of ADCP, the gate is preferably set on THP-1 cells and medianfluorescence intensity is measured. The ADCP activity for individualmutants is calculated and reported as a normalized value to the wildtype chMab 4-4-20 obtained. The opsonized particles are added to THP-1cells such that the ratio of the opsonized particles to THP-1 cells is30:1 or 60:1. In most preferred embodiments, the ADCP assay is conductedwith controls, such as E. coli-FITC in medium, E. coli-FITC and THP-1cells (to serve as FcγR-independent ADCP activity), E. coli-FITC, THP-1cells and wild-type 4-4-20 antibody (to serve as FcγR-dependent ADCPactivity), E. coli-FITC, THP-1 cells, 4-4-20 D265A (to serve as thebackground control for FcγR-dependent ADCP activity).

In another embodiment, the molecules of the invention can be assayed forFcγR-mediated ADCC activity in effector cells, e.g., natural killercells, using any of the standard methods known to those skilled in theart (See e.g., Perussia et al. (2000) “Assays For Antibody-DependentCell-Mediated Cytotoxicity (ADCC) And Reverse ADCC (RedirectedCytotoxicity) In Human Natural Killer Cells,” Methods Mol. Biol. 121:179-92; Weng et al. (2003) “Two Immunoglobulin G Fragment C ReceptorPolymorphisms Independently Predict Response To Rituximab In PatientsWith Follicular Lymphoma,” J. Clin. Oncol. 21:3940-3947; Ding et al.(1998) “Two Human T Cell Receptors Bind In A Similar Diagonal Mode ToThe HLA-A2/Tax Peptide Complex Using Different TCR Amino Acids,”Immunity 8:403-411). An exemplary assay for determining ADCC activity ofthe molecules of the invention is based on a ⁵¹Cr release assaycomprising of: labeling target cells with [⁵¹Cr]Na₂CrO₄ (thiscell-membrane permeable molecule is commonly used for labeling since itbinds cytoplasmic proteins and although spontaneously released from thecells with slow kinetics, it is released massively following target cellnecrosis); opsonizing the target cells with the molecules of theinvention comprising variant heavy chains; combining the opsonizedradiolabeled target cells with effector cells in a microtitre plate atan appropriate ratio of target cells to effector cells; incubating themixture of cells for 16-18 hours at 37° C.; collecting supernatants; andanalyzing radioactivity. The cytotoxicity of the molecules of theinvention can then be determined, for example using the followingformula: % lysis=(experimental cpm−target leak cpm)/(detergent lysiscpm−target leak cpm)×100%. Alternatively, % lysis=(ADCC−AICC)/(maximumrelease-spontaneous release). Specific lysis can be calculated using theformula: specific lysis=% lysis with the molecules of the invention−%lysis in the absence of the molecules of the invention. A graph can begenerated by varying either the target: effector cell ratio or antibodyconcentration.

Preferably, the effector cells used in the ADCC assays of the inventionare peripheral blood mononuclear cells (PBMC) that are preferablypurified from normal human blood, using standard methods known to oneskilled in the art, e.g., using Ficoll-Paque density gradientcentrifugation. Preferred effector cells for use in the methods of theinvention express different FcγR activating receptors. The inventionencompasses, effector cells, THP-1, expressing FcγRI, FcγRIIA andFcγRIIB, and monocyte derived primary macrophages derived from wholehuman blood expressing both FcγRIIIA and FcγRIIB, to determine if heavychain antibody mutants show increased ADCC activity and phagocytosisrelative to wild type IgG1 antibodies.

The human monocyte cell line, THP-1, activates phagocytosis throughexpression of the high affinity receptor FcγRI and the low affinityreceptor FcγRIIA (Fleit et al. (1991) “The Human Monocyte-Like Cell LineTHP-1 Expresses Fc Gamma RI And Fc Gamma RII,” J. Leuk. Biol. 49:556-565). THP-1 cells do not constitutively express FcγRIIA or FcγRIIB.Stimulation of these cells with cytokines affects the FcR expressionpattern (Pricop et al. (2001) “Differential Modulation Of StimulatoryAnd Inhibitory Fc Gamma Receptors On Human Monocytes By Th1 And Th2Cytokines,” J. of Immunol., 166: 531-537). Growth of THP-1 cells in thepresence of the cytokine IL4 induces FcγRIIB expression and causes areduction in FcγRIIA and FcγRI expression. FcγRIIB expression can alsobe enhanced by increased cell density (Tridandapani et al. (2002)“Regulated Expression And Inhibitory Function Of Fcgamma RIIB In HumanMonocytic Cells,” J. Biol. Chem., 277(7): 5082-5089). In contrast, ithas been reported that IFNγ can lead to expression of FcγRIIIA (Pearseet al. (1993) “Interferon Gamma-Induced Transcription Of TheHigh-Affinity Fc Receptor For IgG Requires Assembly Of A Complex ThatIncludes The 91-kDa Subunit Of Transcription Factor ISGF3,” Proc. Nat.Acad. Sci. USA 90: 4314-4318). The presence or absence of receptors onthe cell surface can be determined by FACS using common methods known toone skilled in the art. Cytokine induced expression of FcγR on the cellsurface provides a system to test both activation and inhibition in thepresence of FcγRIIB. If THP-1 cells are unable to express the FcγRIIBthe invention also encompasses another human monocyte cell line, U937.These cells have been shown to terminally differentiate into macrophagesin the presence of IFNγ and TNF (Koren et al. (1979) “In VitroActivation Of A Human Macrophage-Like Cell Line,” Nature 279: 328-331).

FcγR dependent tumor cell killing is mediated by macrophage and NK cellsin mouse tumor models (Clynes et al. (1998) “Fc Receptors Are RequiredIn Passive And Active Immunity To Melanoma,” Proc. Nat. Acad. Sci. USA95: 652-656). The invention encompasses the use of elutriated monocytesfrom donors as effector cells to analyze the efficiency Fc mutants totrigger cell cytotoxicity of target cells in both phagocytosis and ADCCassays. Expression patterns of FcγRI, FcγRIIIA, and FcγRIIB are affectedby different growth conditions. FcγR expression from frozen elutriatedmonocytes, fresh elutriated monocytes, monocytes maintained in 10% FBS,and monocytes cultured in FBS+GM-CSF and or in human serum may bedetermined using common methods known to those skilled in the art. Forexample, cells can be stained with FcγR specific antibodies and analyzedby FACS to determine FcR profiles. Conditions that best mimic macrophagein vivo FcγR expression is then used for the methods of the invention.

In some embodiments, the invention encompasses the use of mouse cellsespecially when human cells with the right FcγR profiles are unable tobe obtained. In some embodiments, the invention encompasses the mousemacrophage cell line RAW264.7(ATCC) which can be transfected with humanFcγRIIIA and stable transfectants isolated using methods known in theart, see, e.g., Ralph et al. (1977) “Antibody-Dependent Killing OfErythrocyte And Tumor Targets By Macrophage-Related Cell LinesEnhancement By PPD And LPS,” J. Immunol. 119: 950-4). Transfectants canbe quantitated for FcγRIIIA expression by FACS analysis using routineexperimentation and high expressors can be used in the ADCC assays ofthe invention. In other embodiments, the invention encompasses isolationof spleen peritoneal macrophage expressing human FcγR from knockouttransgenic mice such as those disclosed herein.

Lymphocytes may be harvested from peripheral blood of donors (PBM) usinga Ficoll-Paque gradient (Pharmacia). Within the isolated mononuclearpopulation of cells the majority of the ADCC activity occurs via thenatural killer cells (NK) containing FcγRIIIA but not FcγRIIB on theirsurface. Results with these cells indicate the efficacy of the mutantson triggering NK cell ADCC and establish the reagents to test withelutriated monocytes.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al. (1976) “Human BreastCancer In Culture,” Recent Results Cancer Res. 33-41); B-lymphocytes;cells derived from Burkitts lymphoma, e.g., Raji cells with ATCCaccession number. CCL-86 (see, e.g., Epstein et al. (1965)“Characteristics And Mode Of Growth Of Tissue Culture Strain (EB1) OfHuman Lymphoblasts From Burkitt's Lymphoma,” J. Natl. Cancer Inst. 34:231-240), and Daudi cells with ATCC accession number CCL-213 (see, e.g.,Klein et al. (1968) “Surface IgM-Kappa Specificity On A Burkitt LymphomaCell In Vivo And In Derived Culture Lines,” Cancer Res. 28: 1300-1310).The target cells must be recognized by the antigen binding site of thediabody molecule to be assayed.

The ADCC assay is based on the ability of NK cells to mediate cell deathvia an apoptotic pathway. NK cells mediate cell death in part byFcγRIIIA's recognition of an IgG Fc domain bound to an antigen on a cellsurface. The ADCC assays used in accordance with the methods of theinvention may be radioactive based assays or fluorescence based assays.The ADCC assay used to characterize the molecules of the inventioncomprising variant Fc regions comprises labeling target cells, e.g.,SK-BR-3, MCF-7, OVCAR3, Raji, Daudi cells, opsonizing target cells withan antibody that recognizes a cell surface receptor on the target cellvia its antigen binding site; combining the labeled opsonized targetcells and the effector cells at an appropriate ratio, which can bedetermined by routine experimentation; harvesting the cells; detectingthe label in the supernatant of the lysed target cells, using anappropriate detection scheme based on the label used. The target cellsmay be labeled either with a radioactive label or a fluorescent label,using standard methods known in the art. For example the labels include,but are not limited to, [⁵¹Cr]Na₂CrO₄; and the acetoxymethyl ester ofthe fluorescence enhancing ligand,2,2′:6′,2″-terpyridine-6-6″-dicarboxylate (TDA).

In a specific preferred embodiment, a time resolved fluorimetric assayis used for measuring ADCC activity against target cells that have beenlabeled with the acetoxymethyl ester of the fluorescence enhancingligand, 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate (TDA). Suchfluorimetric assays are known in the art, e.g., see, Blomberg et al.(1996) “Time-Resolved Fluorometric Assay For Natural Killer ActivityUsing Target Cells Labelled With A Fluorescence Enhancing Ligand,”Journal of Immunological Methods, 193: 199-206; which is incorporatedherein by reference in its entirety. Briefly, target cells are labeledwith the membrane permeable acetoxymethyl diester of TDA(bis(acetoxymethyl) 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate, (BATDA),which rapidly diffuses across the cell membrane of viable cells.Intracellular esterases split off the ester groups and the regeneratedmembrane impermeable TDA molecule is trapped inside the cell. Afterincubation of effector and target cells, e.g., for at least two hours,up to 3.5 hours, at 37° C., under 5% CO₂, the TDA released from thelysed target cells is chelated with Eu3+ and the fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallace).

In another specific embodiment, the ADCC assay used to characterize themolecules of the invention comprising multiple epitope binding sitesand, optionally, an Fc domain (or portion thereof) comprises thefollowing steps: Preferably 4−5×10⁶ target cells (e.g., SK-BR-3, MCF-7,OVCAR3, Raji cells) are labeled with bis(acetoxymethyl)2,2′:6′,2″-terpyridine-t-6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). For optimal labeling efficiency, the number of targetcells used in the ADCC assay should preferably not exceed 5×10⁶. BATDAreagent is added to the cells and the mixture is incubated at 37° C.preferably under 5% CO₂, for at least 30 minutes. The cells are thenwashed with a physiological buffer, e.g., PBS with 0.125 mMsulfinpyrazole, and media containing 0.125 mM sulfinpyrazole. Thelabeled target cells are then opsonized (coated) with a molecule of theinvention comprising an epitope binding domain specific for FcγRIIA and,optionally, an Fc domain (or portion thereof). In preferred embodiments,the molecule used in the ADCC assay is also specific for a cell surfacereceptor, a tumor antigen, or a cancer antigen. The diabody molecule ofthe invention may specifically bind any cancer or tumor antigen, such asthose listed in section 5.6.1. The target cells in the ADCC assay arechosen according to the epitope binding sites engineered into thediabody of the invention, such that the diabody binds a cell surfacereceptor of the target cell specifically.

Target cells are added to effector cells, e.g., PBMC, to produceeffector:target ratios of approximately 1:1, 10:1, 30:1, 50:1, 75:1, or100:1. The effector and target cells are incubated for at least twohours, up to 3.5 hours, at 37° C., under 5% CO₂. Cell supernatants areharvested and added to an acidic europium solution (e.g., DELFIAEuropium Solution, Perkin Elmer/Wallac). The fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallac). Maximal release(MR) and spontaneous release (SR) are determined by incubation of targetcells with 1% TX-100 and media alone, respectively. Antibody independentcellular cytotoxicity (AICC) is measured by incubation of target andeffector cells in the absence of a test molecule, e.g., diabody of theinvention. Each assay is preferably performed in triplicate. The meanpercentage specific lysis is calculated as: Experimental release(ADCC)−AICC)/(MR-SR)×100.

The invention encompasses assays known in the art, and exemplifiedherein, to characterize the binding of C1q and mediation of complementdependent cytotoxicity (CDC) by molecules of the invention comprising Fcdomains (or portions thereof). To determine C1 q binding, a C1q bindingELISA may be performed. An exemplary assay may comprise the following:assay plates may be coated overnight at 4 C with polypeptide comprisinga molecule of the invention or starting polypeptide (control) in coatingbuffer. The plates may then be washed and blocked. Following washing, analiquot of human C1q may be added to each well and incubated for 2 hrsat room temperature. Following a further wash, 100 uL of a sheepanti-complement C1q peroxidase conjugated antibody may be added to eachwell and incubated for 1 hour at room temperature. The plate may againbe washed with wash buffer and 100 ul of substrate buffer containing OPD(O-phenylenediamine dihydrochloride (Sigma)) may be added to each well.The oxidation reaction, observed by the appearance of a yellow color,may be allowed to proceed for 30 minutes and stopped by the addition of100 ul of 4.5 NH2 SO4. The absorbance may then read at (492-405) nm.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, e.g. as described in Gazzano-Santoro etal. (1997) “A Non-Radioactive Complement-Dependent Cytotoxicity AssayFor Anti-CD20 Monoclonal Antibody,” J. Immunol. Methods 202:163-171,which is incorporated herein by reference in its entirety. Briefly,various concentrations of the molecule comprising a (variant) Fc domain(or portion thereof) and human complement may be diluted with buffer.Cells which express the antigen to which the diabody molecule binds maybe diluted to a density of about 1×10⁶ cells/ml. Mixtures of the diabodymolecules comprising a (variant) Fc domain (or portion thereof), dilutedhuman complement and cells expressing the antigen may be added to a flatbottom tissue culture 96 well plate and allowed to incubate for 2 hrs at37° C. and 5% CO₂ to facilitate complement mediated cell lysis. 50 uL ofalamar blue (Accumed International) may then be added to each well andincubated overnight at 37° C. The absorbance is measured using a 96-wellfluorometer with excitation at 530 nm and emission at 590 nm. Theresults may be expressed in relative fluorescence units (RFU). Thesample concentrations may be computed from a standard curve and thepercent activity as compared to nonvariant molecule, i.e., a moleculenot comprising an Fc domain or comprising a non-variant Fc domain, isreported for the variant of interest.

5.4.3 Other Assays

The molecules of the invention comprising multiple epitope bindingdomain and, optionally, an Fc domain may be assayed using any surfaceplasmon resonance based assays known in the art for characterizing thekinetic parameters of an antigen-binding domain or Fc-FcγR binding. AnySPR instrument commercially available including, but not limited to,BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsysinstruments available from Affinity Sensors (Franklin, Mass.); IBISsystem available from Windsor Scientific Limited (Berks, UK), SPR-CELLIAsystems available from Nippon Laser and Electronics Lab (Hokkaido,Japan), and SPR Detector Spreeta available from Texas Instruments(Dallas, Tex.) can be used in the instant invention. For a review ofSPR-based technology see Mullet et al. (2000) “Surface PlasmonResonance-Based Immunoassays,” Methods 22: 77-91; Dong et al. (2002)“Some new aspects in biosensors,” Reviews in Mol. Biotech. 82: 303-23;Fivash et al. (1998) “BIAcore For Macromolecular Interaction,” CurrentOpinion in Biotechnology 9: 97-101; Rich et al. (2000) “Advances InSurface Plasmon Resonance Biosensor Analysis,” Current Opinion inBiotechnology 11: 54-61; all of which are incorporated herein byreference in their entirety. Additionally, any of the SPR instrumentsand SPR based methods for measuring protein-protein interactionsdescribed in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215;6,268,125, all of which are incorporated herein by reference in theirentirety, are contemplated in the methods of the invention.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occurs is the sensor chip, which is at the heart ofthe SPR technology; it consists of a glass surface coated with a thinlayer of gold and forms the basis for a range of specialized surfacesdesigned to optimize the binding of a molecule to the surface. A varietyof sensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to, direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups, and attachmentthrough the histidine tag with NTA chips.

In some embodiments, the kinetic parameter's of the binding of moleculesof the invention comprising multiple epitope binding sites and,optionally, and Fc domain, to an antigen or an FcγR may be determinedusing a BIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc.,Piscataway, N.J.). As discussed supra, see section 5.4.1, any FcγR canbe used to assess the binding of the molecules of the invention eitherwhere at least one epitope binding site of the diabody moleculeimmunospecifically recognizes an FcγR, and/or where the diabody moleculecomprises an Fc domain (or portion thereof). In a specific embodimentthe FcγR is FcγRIIIA, preferably a soluble monomeric FcγRIIIA. Forexample, in one embodiment, the soluble monomeric FcγRIIIA is theextracellular region of FcγRIIIA joined to the linker-AVITAG sequence(see, U.S. Provisional Application No. 60/439,498, filed on Jan. 9, 2003and U.S. Provisional Application No. 60/456,041 filed on Mar. 19, 2003,which are incorporated herein by reference in their entireties). Inanother specific embodiment, the FcγR is FcγRIIB, preferably a solubledimeric FcγRIIB. For example in one embodiment, the soluble dimericFcγRIIB protein is prepared in accordance with the methodology describedin U.S. Provisional application No. 60/439,709 filed on Jan. 13, 2003,which is incorporated herein by reference in its entirety.

For all immunological assays, FcγR recognition/binding by a molecule ofthe invention may be effected by multiple domains: in certainembodiments, molecules of the invention immunospecifically recognize anFcγR via one of the multiple epitope binding domains; in yet otherembodiments, where the molecule of the invention comprises an Fc domain(or portion thereof), the diabody molecule may immunospecificallyrecognize an FcγR via Fc-FcγR interactions; in yet further embodiments,where a molecule of the invention comprises both an Fc domain (orportion thereof) and an epitope binding site that immunospecificallyrecognizes an FcγR, the diabody molecule may recognize an FcγR via oneor both of an epitope binding domain and the Fc domain (or portionthereof). An exemplary assay for determining the kinetic parameters of amolecule comprising multiple epitope binding domains and, optionally,and Fc domain (or portion thereof) to an antigen and/or an FcγR using aBIAcore instrument comprises the following: a first antigen isimmobilized on one of the four flow cells of a sensor chip surface,preferably through amine coupling chemistry such that about 5000response units (RU) of said first antigen is immobilized on the surface.Once a suitable surface is prepared, molecules of the invention thatimmunospecifically recognize said first antigen are passed over thesurface, preferably by one minute injections of a 20 μg/mL solution at a5 μL/mL flow rate. Levels of molecules of the invention bound to thesurface at this stage typically ranges between 400 and 700 RU. Next,dilution series of a second antigen (e.g., FcγR) or FcγR receptor inHBS-P buffer (20 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.5) are injectedonto the surface at 100 μL/min Regeneration of molecules betweendifferent second antigen or receptor dilutions is carried out preferablyby single 5 second injections of 100 mM NaHCO₃ pH 9.4; 3M NaCl. Anyregeneration technique known in the art is contemplated in the method ofthe invention.

Once an entire data set is collected, the resulting binding curves areglobally fitted using computer algorithms supplied by the SPR instrumentmanufacturer, e.g., BIAcore, Inc. (Piscataway, N.J.). These algorithmscalculate both the K_(on) and K_(off), from which the apparentequilibrium binding constant, IQ is deduced as the ratio of the two rateconstants (i.e., K_(off)/K_(on)). More detailed treatments of how theindividual rate constants are derived can be found in the BIAevaluaionSoftware Handbook (BIAcore, Inc., Piscataway, N.J.). The analysis of thegenerated data may be done using any method known in the art. For areview of the various methods of interpretation of the kinetic datagenerated see Myszka (1997) “Kinetic Analysis Of MacromolecularInteractions Using Surface Plasmon Resonance Biosensors,” CurrentOpinion in Biotechnology 8: 50-7; Fisher et al. (1994) “Surface PlasmonResonance Based Methods For Measuring The Kinetics And BindingAffinities Of Biomolecular Interactions,” Current Opinion inBiotechnology 5: 389-95; O'Shannessy (1994) “Determination Of KineticRate And Equilibrium Binding Constants For Macromolecular Interactions:A Critique Of The Surface Plasmon Resonance Literature,” Current Opinionin Biotechnology, 5:65-71; Chaiken et al. (1992) “Analysis OfMacromolecular Interactions Using Immobilized Ligands,” AnalyticalBiochemistry, 201: 197-210; Morton et al. (1995) “Interpreting ComplexBinding Kinetics From Optical Biosensors: A Comparison Of Analysis ByLinearization, The Integrated Rate Equation, And Numerical Integration,”Analytical Biochemistry 227: 176-85; O'Shannessy et al., 1996,Analytical Biochemistry 236: 275-83; all of which are incorporatedherein by reference in their entirety.

In preferred embodiments, the kinetic parameters determined using an SPRanalysis, e.g., BIAcore, may be used as a predictive measure of how amolecule of the invention will function in a functional assay, e.g.,ADCC. An exemplary method for predicting the efficacy of a molecule ofthe invention based on kinetic parameters obtained from an SPR analysismay comprise the following: determining the K_(off) values for bindingof a molecule of the invention to FcγRIIIA and FcγRIIB (via an epitopebinding domain and/or an Fc domain (or portion thereof)); plotting (1)K_(off)(wt)/K_(off) (mut) for FcγRIIIA; (2) K_(off) (mut)/K_(off) (wt)for FcγRIIB against the ADCC data. Numbers higher than one show adecreased dissociation rate for FcγRIIIA and an increased dissociationrate for FcγRIIB relative to wild type; and possess and enhanced ADCCfunction.

5.5 Methods of Producing Diabody Molecules of the Invention

The diabody molecules of the present invention can be produced using avariety of methods well known in the art, including de novo proteinsynthesis and recombinant expression of nucleic acids encoding thebinding proteins. The desired nucleic acid sequences can be produced byrecombinant methods (e.g., PCR mutagenesis of an earlier preparedvariant of the desired polynucleotide) or by solid-phase DNA synthesis.Usually recombinant expression methods are used. In one aspect, theinvention provides a polynucleotide that comprises a sequence encoding aCD16A VH and/or VL; in another aspect, the invention provides apolynucleotide that comprises a sequence encoding a CD32B VH and/or VL.Because of the degeneracy of the genetic code, a variety of nucleic acidsequences encode each immunoglobulin amino acid sequence, and thepresent invention includes all nucleic acids encoding the bindingproteins described herein.

5.5.1 Polynucleotides Encoding Molecules of the Invention

The present invention also includes polynucleotides that encode themolecules of the invention, including the polypeptides and antibodies.The polynucleotides encoding the molecules of the invention may beobtained, and the nucleotide sequence of the polynucleotides determined,by any method known in the art.

Once the nucleotide sequence of the molecules that are identified by themethods of the invention is determined, the nucleotide sequence may bemanipulated using methods well known in the art, e.g., recombinant DNAtechniques, site directed mutagenesis, PCR, etc. (see, for example, thetechniques described in Sambrook et al., 2001, Molecular Cloning, ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; and Ausubel et al., eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY, which are both incorporated byreference herein in their entireties), to generate, for example,antibodies having a different amino acid sequence, for example bygenerating amino acid substitutions, deletions, and/or insertions.

In one embodiment, human libraries or any other libraries available inthe art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the molecules of the invention.

5.5.2 Recombinant Expression of Molecules of the Invention

Once a nucleic acid sequence encoding molecules of the invention (i.e.,antibodies) has been obtained, the vector for the production of themolecules may be produced by recombinant DNA technology using techniqueswell known in the art. Methods which are well known to those skilled inthe art can be used to construct expression vectors containing thecoding sequences for the molecules of the invention and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, for example, thetechniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of a moleculeidentified by the methods of the invention can be transferred to a hostcell by conventional techniques (e.g., electroporation, liposomaltransfection, and calcium phosphate precipitation) and the transfectedcells are then cultured by conventional techniques to produce themolecules of the invention. In specific embodiments, the expression ofthe molecules of the invention is regulated by a constitutive, aninducible or a tissue, specific promoter.

The host cells used to express the molecules identified by the methodsof the invention may be either bacterial cells such as Escherichia coli,or, preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells,such as Chinese hamster ovary cells (CHO), in conjunction with a vectorsuch as the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al. (1986) “Powerful And Versatile Enhancer-Promoter UnitFor Mammalian Expression Vectors,” Gene 45:101-106; Cockett et al.(1990) “High Level Expression Of Tissue Inhibitor Of MetalloproteinasesIn Chinese Hamster Ovary Cells Using Glutamine Synthetase GeneAmplification,” Biotechnology 8:662-667).

A variety of host-expression vector systems may be utilized to expressthe molecules identified by the methods of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof the molecules of the invention may be produced and subsequentlypurified, but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, expressthe molecules of the invention in situ. These include, but are notlimited to, microorganisms such as bacteria (e.g., E. coli and B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing coding sequences for themolecules identified by the methods of the invention; yeast (e.g.,Saccharomyces pichia) transformed with recombinant yeast expressionvectors containing sequences encoding the molecules identified by themethods of the invention; insect cell systems infected with recombinantvirus expression vectors (e.g., baclovirus) containing the sequencesencoding the molecules identified by the methods of the invention; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing sequences encoding the molecules identified by themethods of the invention; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (human retinal cells developed by Crucell)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the moleculebeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Rüther et al. (1983) “Easy Identification Of cDNA Clones,” EMBOJ. 2:1791-1794), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye et al. (1985)“Up-Promoter Mutations In The lpp Gene Of Escherichia Coli,” NucleicAcids Res. 13:3101-3110; Van Heeke et al. (1989) “Expression Of HumanAsparagine Synthetase In Escherichia Coli,” J. Biol. Chem.24:5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrixglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts (e.g., seeLogan et al. (1984) “Adenovirus Tripartite Leader Sequence EnhancesTranslation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al. (1987) “Expression And Secretion Vectors For Yeast,”Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. For example, in certainembodiments, the polypeptides comprising a diabody molecule of theinvention may be expressed as a single gene product (e.g., as a singlepolypeptide chain, i.e., as a polyprotein precursor), requiringproteolytic cleavage by native or recombinant cellular mechanisms toform the separate polypeptides of the diabody molecules of theinvention. The invention thus encompasses engineering a nucleic acidsequence to encode a polyprotein precursor molecule comprising thepolypeptides of the invention, which includes coding sequences capableof directing post translational cleavage of said polyprotein precursor.Post-translational cleavage of the polyprotein precursor results in thepolypeptides of the invention. The post translational cleavage of theprecursor molecule comprising the polypeptides of the invention mayoccur in vivo (i.e., within the host cell by native or recombinant cellsystems/mechanisms, e.g. furin cleavage at an appropriate site) or mayoccur in vitro (e.g. incubation of said polypeptide chain in acomposition comprising proteases or peptidases of known activity and/orin a composition comprising conditions or reagents known to foster thedesired proteolytic action). Purification and modification ofrecombinant proteins is well known in the art such that the design ofthe polyprotein precursor could include a number of embodiments readilyappreciated by a skilled worker. Any known proteases or peptidases knownin the art can be used for the described modification of the precursormolecule, e.g., thrombin (which recognizes the amino acid sequenceLVPR^(̂)GS (SEQ ID NO: 89)), or factor Xa (which recognizes the aminoacid sequence I(E/D)GR^(̂) (SEQ ID NO: 90) (Nagai et al. (1985) “OxygenBinding Properties Of Human Mutant Hemoglobins Synthesized InEscherichia Coli,” Proc. Nat. Acad. Sci. USA 82:7252-7255, and reviewedin Jenny et al. (2003) “A Critical Review Of The Methods For Cleavage OfFusion Proteins With Thrombin And Factor Xa,” Protein Expr. Purif.31:1-11, each of which is incorporated by reference herein in itsentirety)), enterokinase (which recognizes the amino acid sequenceDDDDK^(̂ (SEQ ID NO:) 91) (Collins-Racie et al. (1995) “Production OfRecombinant Bovine Enterokinase Catalytic Subunit In Escherichia ColiUsing The Novel Secretory Fusion Partner DsbA,” Biotechnology 13:982-987hereby incorporated by reference herein in its entirety)), furin (whichrecognizes the amino acid sequence RXXR^(̂), with a preference forRX(K/R)R^(̂) (SEQ ID NO: 92 and SEQ ID NO: 93, respectively) (additionalR at P6 position appears to enhance cleavage)), and AcTEV (whichrecognizes the amino acid sequence ENLYFQ^(̂)G (SEQ ID NO: 94) (Parks etal. (1994) “Release Of Proteins And Peptides From Fusion Proteins UsingA Recombinant Plant Virus Proteinase,” Anal. Biochem. 216:413-417 herebyincorporated by reference herein in its entirety)) and the Foot andMouth Disease Virus Protease C3. See for example, section 6.4, supra.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 andT47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the molecules of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al. (1977)“Transfer Of Purified Herpes Virus Thymidine Kinase Gene To CulturedMouse Cells,” Cell 11: 223-232), hypoxanthine-guaninephosphoribosyltransferase (Szybalska et al. (1992) “Use Of The HPRT GeneAnd The HAT Selection Technique In DNA-Mediated Transformation OfMammalian Cells First Steps Toward Developing Hybridoma Techniques AndGene Therapy,” Bioessays 14: 495-500), and adeninephosphoribosyltransferase (Lowy et al. (1980) “Isolation Of TransformingDNA: Cloning The Hamster aprt Gene,” Cell 22: 817-823) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al. (1980) “Transformation Of Mammalian Cells With An AmplifiableDominant-Acting Gene,” Proc. Natl. Acad. Sci. USA 77:3567-3570; O'Hareet al. (1981) “Transformation Of Mouse Fibroblasts To MethotrexateResistance By A Recombinant Plasmid Expressing A ProkaryoticDihydrofolate Reductase,” Proc. Natl. Acad. Sci. USA 78: 1527-1531);gpt, which confers resistance to mycophenolic acid (Mulligan et al.(1981) “Selection For Animal Cells That Express The Escherichia coliGene Coding For Xanthine-Guanine Phosphoribosyltransferase,” Proc. Natl.Acad. Sci. USA 78: 2072-2076); neo, which confers resistance to theaminoglycoside G-418 (Tolstoshev (1993) “Gene Therapy, Concepts, CurrentTrials And Future Directions,” Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan (1993) “The Basic Science Of Gene Therapy,” Science260:926-932; and Morgan et al. (1993) “Human Gene Therapy,” Ann. Rev.Biochem. 62:191-217) and hygro, which confers resistance to hygromycin(Santerre et al. (1984) “Expression Of Prokaryotic Genes For HygromycinB And G418 Resistance As Dominant-Selection Markers In Mouse L Cells,”Gene 30:147-156). Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY;Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds),1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.;Colberre-Garapin et al. (1981) “A New Dominant Hybrid Selective MarkerFor Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14.

The expression levels of a molecule of the invention can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987). When a marker in the vector system expressing an antibodyis amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of apolypeptide of the diabody molecule, production of the polypeptide willalso increase (Crouse et al. (1983) “Expression And Amplification OfEngineered Mouse Dihydrofolate Reductase Minigenes,” Mol. Cell. Biol.3:257-266).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding the first polypeptide of thediabody molecule and the second vector encoding the second polypeptideof the diabody molecule. The two vectors may contain identicalselectable markers which enable equal expression of both polypeptides.Alternatively, a single vector may be used which encodes bothpolypeptides. The coding sequences for the polypeptides of the moleculesof the invention may comprise cDNA or genomic DNA.

Once a molecule of the invention (i.e., diabodies) has beenrecombinantly expressed, it may be purified by any method known in theart for purification of polypeptides, polyproteins or diabodies (e.g.,analogous to antibody purification schemes based on antigen selectivity)for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen (optionally afterProtein A selection where the diabody molecule comprises an Fc domain(or portion thereof)), and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of polypeptides, polyproteins ordiabodies.

5.6 Prophylactic and Therapeutic Methods

The molecules of the invention are particularly useful for the treatmentand/or prevention of a disease, disorder or infection where an effectorcell function (e.g., ADCC) mediated by FcγR is desired (e.g., cancer,infectious disease). As discussed supra, the diabodies of the inventionmay exhibit antibody-like functionality in eliciting effector functionalthough the diabody molecule does not comprise and Fc domain. Bycomprising at least one epitope binding domain that immunospecificallyrecognizes an FcγR, the diabody molecule may exhibit FcγR binding andactivity analogous to Fc-FcγR interactions. For example, molecules ofthe invention may bind a cell surface antigen and an FcγR (e.g.,FcγRIIIA) on an immune effector cell (e.g., NK cell), stimulating aneffector function (e.g., ADCC, CDC, phagocytosis, opsonization, etc.)against said cell.

In other embodiments, the diabody molecule of the invention comprises anFc domain (or portion thereof). In such embodiments, the Fc domain mayfurther comprise at least one amino acid modification relative to awild-type Fc domain (or portion thereof) and/or may comprise domainsfrom one or more IgG isotypes (e.g., IgG1, IgG2, IgG3 or IgG4).Molecules of the invention comprising variant Fc domains may exhibitconferred or altered phenotypes relative to molecules comprising thewild type Fc domain such as an altered or conferred effector functionactivity (e.g., as assayed in an NK dependent or macrophage dependentassay). In said embodiments, molecules of the invention with conferredor altered effector function activity are useful for the treatmentand/or prevention of a disease, disorder or infection where an enhancedefficacy of effector function activity is desired. In certainembodiments, the diabody molecules of the invention comprising an Fcdomain (or portion thereof) mediate complement dependent cascade. Fcdomain variants identified as altering effector function are disclosedin International Application WO04/063351, U.S. Patent ApplicationPublications 2005/0037000 and 2005/0064514, U.S. ProvisionalApplications 60/626,510, filed Nov. 10, 2004, 60/636,663, filed Dec. 15,2004, and 60/781,564, filed Mar. 10, 2006, and U.S. patent applicationSer. Nos. 11/271,140, filed Nov. 10, 2005, and 11/305,787, filed Dec.15, 2005, concurrent applications of the Inventors, each of which isincorporated by reference in its entirety.

The invention encompasses methods and compositions for treatment,prevention or management of a cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of oneor more molecules comprising one or more epitope binding sites, andoptionally, an Fc domain (or portion thereof) engineered in accordancewith the invention, which molecule further binds a cancer antigen.Molecules of the invention are particularly useful for the prevention,inhibition, reduction of growth or regression of primary tumors,metastasis of cancer cells, and infectious diseases. Although notintending to be bound by a particular mechanism of action, molecules ofthe invention mediate effector function resulting in tumor clearance,tumor reduction or a combination thereof. In alternate embodiments, thediabodies of the invention mediate therapeutic activity by cross-linkingof cell surface antigens and/or receptors and enhanced apoptosis ornegative growth regulatory signaling.

Although not intending to be bound by a particular mechanism of action,the diabody molecules of the invention exhibit enhanced therapeuticefficacy relative to therapeutic antibodies known in the art, in part,due to the ability of diabody to immunospecifically bind a target cellwhich expresses a particular antigen (e.g., FcγR) at reduced levels, forexample, by virtue of the ability of the diabody to remain on the targetcell longer due to an improved avidity of the diabody-epitopeinteraction.

The diabodies of the invention with enhanced affinity and avidity forantigens (e.g., FcγR5) are particularly useful for the treatment,prevention or management of a cancer, or another disease or disorder, ina subject, wherein the FcγR5 are expressed at low levels in the targetcell populations. As used herein, FcγR expression in cells is defined interms of the density of such molecules per cell as measured using commonmethods known to those skilled in the art. The molecules of theinvention comprising multiple epitope binding sites and, optionally, andFcγR (or portion thereof) preferably also have a conferred or anenhanced avidity and affinity and/or effector function in cells whichexpress a target antigen, e.g., a cancer antigen, at a density of 30,000to 20,000 molecules/cell, at a density of 20,000 to 10,000molecules/cell, at a density of 10,000 molecules/cell or less, at adensity of 5000 molecules/cell or less, or at a density of 1000molecules/cell or less. The molecules of the invention have particularutility in treatment, prevention or management of a disease or disorder,such as cancer, in a sub-population, wherein the target antigen isexpressed at low levels in the target cell population.

The molecules of the invention may also be advantageously utilized incombination with other therapeutic agents known in the art for thetreatment or prevention of diseases, such as cancer, autoimmune disease,inflammatory disorders, and infectious diseases. In a specificembodiment, molecules of the invention may be used in combination withmonoclonal or chimeric antibodies, lymphokines, or hematopoietic growthfactors (such as, e.g., IL-2, IL-3 and IL-7), which, for example, serveto increase the number or activity of effector cells which interact withthe molecules and, increase immune response. The molecules of theinvention may also be advantageously utilized in combination with one ormore drugs used to treat a disease, disorder, or infection such as, forexample anti-cancer agents, anti-inflammatory agents or anti-viralagents, e.g., as detailed in Section 5.7.

5.6.1 Cancers

The invention encompasses methods and compositions for treatment orprevention of cancer in a subject comprising administering to thesubject a therapeutically effective amount of one or more moleculescomprising multiple epitope binding domains. In some embodiments, theinvention encompasses methods and compositions for the treatment orprevention of cancer in a subject with FcγR polymorphisms such as thosehomozygous for the FγRIIIA-158V or FcγRIIIA-158F alleles. In someembodiments, the invention encompasses engineering at least one epitopebinding domain of the diabody molecule to immunospecifically bindFcγRIIIA (158F). In other embodiments, the invention encompassesengineering at least one epitope binding domain of the diabody moleculeto immunospecifically bind FcγRIIIA (158V).

The efficacy of standard monoclonal antibody therapy depends on the FcγRpolymorphism of the subject (Cartron et al. (2002) “Therapeutic ActivityOf Humanized Anti-CD20 Monoclonal Antibody And Polymorphism In IgG FcReceptor FcRIIIa Gene,” Blood 99: 754-758; Weng et al. (2003) “TwoImmunoglobulin G Fragment C Receptor Polymorphisms Independently PredictResponse To Rituximab In Patients With Follicular Lymphoma,” J ClinOncol. 21(21):3940-3947, both of which are incorporated herein byreference in their entireties). These receptors are expressed on thesurface of the effector cells and mediate ADCC. High affinity alleles,of the low affinity activating receptors, improve the effector cells'ability to mediate ADCC. In contrast to relying on Fc-FcγR interactionsto effect effector function, the methods of the invention encompassengineering molecules to immunospecifically recognize the low affinityactivating receptors, allowing the molecules to be designed for aspecific polymorphism. Alternately or additionally, the molecule of theinvention may be engineered to comprise a variant Fc domain thatexhibits enhanced affinity to FcγR (relative to a wild type Fc domain)on effector cells. The engineered molecules of the invention providebetter immunotherapy reagents for patients regardless of their FcγRpolymorphism.

Diabody molecules engineered in accordance with the invention are testedby ADCC using either a cultured cell line or patient derived PMBC cellsto determine the ability of the Fc mutations to enhance ADCC. StandardADCC is performed using methods disclosed herein. Lymphocytes areharvested from peripheral blood using a Ficoll-Paque gradient(Pharmacia). Target cells, i.e., cultured cell lines or patient derivedcells, are loaded with Europium (PerkinElmer) and incubated witheffectors for 4 hrs at 37° C. Released Europium is detected using afluorescent plate reader (Wallac). The resulting ADCC data indicates theefficacy of the molecules of the invention to trigger NK cell mediatedcytotoxicity and establish which molecules can be tested with bothpatient samples and elutriated monocytes. Diabody molecules showing thegreatest potential for eliciting ADCC activity are then tested in anADCC assay using PBMCs from patients. PBMC from healthy donors are usedas effector cells.

Accordingly, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen by engineering the diabodymolecule to immunospecifically recognize said cancer antigen such thatthe diabody molecule is itself cytotoxic (e.g., via crosslinking ofsurface receptors leading to increased apoptosis or downregulation ofproliferative signals) and/or comprises an Fc domain, according to theinvention, and/or mediates one or more effector function (e.g., ADCC,phagocytosis). The diabodies that have been engineered according to theinvention are useful for prevention or treatment of cancer, since theyhave an cytotoxic activity (e.g., enhanced tumor cell killing and/orenhanced for example, ADCC activity or CDC activity).

Cancers associated with a cancer antigen may be treated or prevented byadministration of a diabody that binds a cancer antigen and iscytotoxic, and/or has been engineered according to the methods of theinvention to exhibit effector function. For example, but not by way oflimitation, cancers associated with the following cancer antigens may betreated or prevented by the methods and compositions of the invention:KS 1/4 pan-carcinoma antigen (Perez et al. (1989) “Isolation AndCharacterization Of A Cdna Encoding The Ks1/4 Epithelial CarcinomaMarker,” J. Immunol. 142:3662-3667; Möller et al. (1991)“Bispecific-Monoclonal-Antibody-Directed Lysis Of Ovarian CarcinomaCells By Activated Human T Lymphocytes,” Cancer Immunol. Immunother.33(4):210-216), ovarian carcinoma antigen (CA125) (Yu et al. (1991)“Coexpression Of Different Antigenic Markers On Moieties That Bear CA125 Determinants,” Cancer Res. 51(2):468-475), prostatic acid phosphate(Tailor et al. (1990) “Nucleotide Sequence Of Human Prostatic AcidPhosphatase Determined From A Full-Length cDNA Clone,” Nucl. Acids Res.18(16):4928), prostate specific antigen (Henttu et al. (1989) “cDNACoding For The Entire Human Prostate Specific Antigen Shows HighHomologies To The Human Tissue Kallikrein Genes,” Biochem. Biophys. Res.Comm. 10(2):903-910; Israeli et al. (1993) “Molecular Cloning Of AComplementary DNA Encoding A Prostate-Specific Membrane Antigen,” CancerRes. 53:227-230), melanoma-associated antigen p97 (Estin et al. (1989)“Transfected Mouse Melanoma Lines That Express Various Levels Of HumanMelanoma-Associated Antigen p97,” J. Natl. Cancer Instit.81(6):445-454), melanoma antigen gp75 (Vijayasardahl et al. (1990) “TheMelanoma Antigen Gp75 Is The Human Homologue Of The Mouse B (Brown)Locus Gene Product,” J. Exp. Med. 171(4):1375-1380), high molecularweight melanoma antigen (HMW-MAA) (Natali et al. (1987)“Immunohistochemical Detection Of Antigen In Human Primary AndMetastatic Melanomas By The Monoclonal Antibody 140.240 And Its PossiblePrognostic Significance,” Cancer 59:55-63; Mittelman et al. (1990)“Active Specific Immunotherapy In Patients With Melanoma. A ClinicalTrial With Mouse Antiidiotypic Monoclonal Antibodies Elicited WithSyngeneic Anti-High-Molecular-Weight-Melanoma-Associated AntigenMonoclonal Antibodies,” J. Clin. Invest. 86:2136-2144)), prostatespecific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al.(1995) “Immune Response To The Carcinoembryonic Antigen In PatientsTreated With An Anti-Idiotype Antibody Vaccine,” J. Clin. Invest.96(1):334-42), polymorphic epithelial mucin antigen, human milk fatglobule antigen, Colorectal tumor-associated antigens such as: CEA,TAG-72 (Yokota et al. (1992) “Rapid Tumor Penetration Of A Single-ChainFv And Comparison With Other Immunoglobulin Forms,” Cancer Res.52:3402-3408), CO17-1A (Ragnhammar et al. (1993) “Effect Of MonoclonalAntibody 17-1A And GM-CSF In Patients With Advanced ColorectalCarcinoma—Long-Lasting, Complete Remissions Can Be Induced,” Int. J.Cancer 53:751-758); GICA 19-9 (Herlyn et al. (1982) “Monoclonal AntibodyDetection Of A Circulating Tumor-Associated Antigen. I. Presence OfAntigen In Sera Of Patients With Colorectal, Gastric, And PancreaticCarcinoma,” J. Clin. Immunol. 2:135-140), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al. (1994) “Anti-CD19 InhibitsThe Growth Of Human B-Cell Tumor Lines In Vitro And Of Daudi Cells InSCID Mice By Inducing Cell Cycle Arrest,” Blood 83:1329-1336), humanB-lymphoma antigen-CD20 (Reff et al. (1994) “Depletion Of B Cells InVivo By A Chimeric Mouse Human Monoclonal Antibody To CD20,” Blood83:435-445), CD33 (Sgouros et al. (1993) “Modeling And Dosimetry OfMonoclonal Antibody M195 (Anti-CD33) In Acute Myelogenous Leukemia,” J.Nucl. Med. 34:422-430), melanoma specific antigens such as gangliosideGD2 (Saleh et al. (1993) “Generation Of A Human Anti-Idiotypic AntibodyThat Mimics The GD2 Antigen,” J. Immunol., 151, 3390-3398), gangliosideGD3 (shiara et al. (1993) “A Mouse/Human Chimeric Anti-(Ganglioside GD3)Antibody With Enhanced Antitumor Activities,” Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al. (1994)“Improved Survival In Stage III Melanoma Patients With GM2 Antibodies: ARandomized Trial Of Adjuvant Vaccination With GM2 Ganglioside,” J. Clin.Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al. (1993) “MolecularCloning Of A Human Monoclonal Antibody Reactive To Ganglioside GM3Antigen On Human Cancers,” Cancer Res. 53:5244-5250), tumor-specifictransplantation type of cell-surface antigen (TSTA) such asvirally-induced tumor antigens including T-antigen DNA tumor viruses andenvelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellström et al. (1985) “Monoclonal Antibodies To Cell SurfaceAntigens Shared By Chemically Induced Mouse Bladder Carcinomas,” Cancer.Res. 45:2210-2188), differentiation antigen such as human lung carcinomaantigen L6, L20 (Hellström et al. (1986) “Monoclonal Mouse AntibodiesRaised Against Human Lung Carcinoma,” Cancer Res. 46:3917-3923),antigens of fibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al. (1988) “Idiotype Vaccines Against HumanT Cell Leukemia II. Generation And Characterization Of A MonoclonalIdiotype Cascade (Ab1, Ab2, and Ab3),” J. Immunol. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER)2),polymorphic epithelial mucin (PEM) (Hilkens et al. (1992) “CellMembrane-Associated Mucins And Their Adhesion-Modulating Property,”Trends in Biochem. Sci. 17:359-363), malignant human lymphocyteantigen-APO-1 (Trauth et al. (1989) “Monoclonal Antibody-Mediated TumorRegression By Induction Of Apoptosis,” Science 245:301-304),differentiation antigen (Feizi (1985) “Demonstration By MonoclonalAntibodies That Carbohydrate Structures Of Glycoproteins And GlycolipidsAre Onco-Developmental Antigens,” Nature 314:53-57) such as I antigenfound in fetal erthrocytes and primary endoderm, I(Ma) found in gastricadenocarcinomas, M18 and M39 found in breast epithelium, SSEA-1 found inmyeloid cells, VEP8, VEP9, Myl, VIM-D5, and D₁56-22 found in colorectalcancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma,F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten,Le^(y) found in embryonal carcinoma cells, TL5 (blood group A), EGFreceptor found in A431 cells, E₁ series (blood group B) found inpancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastricadenocarcinoma, CO-514 (blood group Le^(a)) found in adenocarcinoma,NS-10 found in adenocarcinomas, CO-43 (blood group Le^(b)), G49, EGFreceptor, (blood group ALe^(b)/Le^(y)) found in colonic adenocarcinoma,19.9 found in colon cancer, gastric cancer mucins, T₅A₇ found in myeloidcells, R₂₄ found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2,G_(D2), M1:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4found in 4-8-cell stage embryos. In another embodiment, the antigen is aT cell receptor derived peptide from a cutaneous T cell lymphoma (seeEdelson (1998) “Cutaneous T-Cell Lymphoma: A Model For SelectiveImmunotherapy,” Cancer J Sci Am. 4:62-71).

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: Leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia Vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungaling(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including but not limitedto, squamous cell cancer, and verrucous; skin cancers including but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including but not limited to, transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcbma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al. (1985) Medicine, 2d Ed., J. B. Lippincott Co.,Philadelphia; and Murphy et al. (1997) Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroidand skin; including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

In a specific embodiment, a molecule of the invention (e.g., a diabodycomprising multiple epitope binding domains and, optionally, and Fcdomain (or portion thereof)) inhibits or reduces the growth of cancercells by at least 99%, at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to the growth ofcancer cells in the absence of said molecule of the invention.

In a specific embodiment, a molecule of the invention (e.g., a diabodycomprising multiple epitope binding domains and, optionally, and Fcdomain (or portion thereof)) kills cells or inhibits or reduces thegrowth of cancer cells at least 5%, at least 10%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 100% better than the parentmolecule.

5.6.2 Autoimmune Disease and Inflammatory Diseases

In some embodiments, molecules of the invention comprise an epitopebinding domain specific for FcγRIIB and or/a variant Fc domain (orportion thereof), engineered according to methods of the invention,which Fc domain exhibits greater affinity for FcγRIIB and decreasedaffinity for FcγRIIIA and/or FcγRIIA relative to a wild-type Fc domain.Molecules of the invention with such binding characteristics are usefulin regulating the immune response, e.g., in inhibiting the immuneresponse in connection with autoimmune diseases or inflammatorydiseases. Although not intending to be bound by any mechanism of action,molecules of the invention with an affinity for FcγRIIB and/orcomprising an Fc domain with increased affinity for FcγRIIB and adecreased affinity for FcγRIIIA and/or FcγRIIA may lead to dampening ofthe activating response to FcγR and inhibition of cellularresponsiveness, and thus have therapeutic efficacy for treating and/orpreventing an autoimmune disorder.

The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an inflammatory disorderin a subject further comprising, administering to said subject atherapeutically or prophylactically effective amount of one or moreanti-inflammatory agents. The invention also provides methods forpreventing, treating, or managing one or more symptoms associated withan autoimmune disease further comprising, administering to said subjecta therapeutically or prophylactically effective amount of one or moreimmunomodulatory agents. Section 5.7 provides non-limiting examples ofanti-inflammatory agents and immunomodulatory agents.

Examples of autoimmune disorders that may be treated by administeringthe molecules of the present invention include, but are not limited to,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Menière's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein inSection 2.2.2, some autoimmune disorders are associated with aninflammatory condition. Thus, there is overlap between what isconsidered an autoimmune disorder and an inflammatory disorder.Therefore, some autoimmune disorders may also be characterized asinflammatory disorders. Examples of inflammatory disorders which can beprevented, treated or managed in accordance with the methods of theinvention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

Molecules of the invention comprising at least one epitope bindingdomain specific for FcγRIIB and/or a variant Fc domain with an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA can also beused to reduce the inflammation experienced by animals, particularlymammals, with inflammatory disorders. In a specific embodiment, amolecule of the invention reduces the inflammation in an animal by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the inflammation in an animal,which is not administered the said molecule.

Molecules of the invention comprising at least one epitope bindingdomain specific for FcγRIIB and/or a variant Fc domain with an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA can also beused to prevent the rejection of transplants.

5.6.3 Infectious Disease

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylatically effective amount of one or moremolecules of the invention comprising at least one epitope bindingdomain specific for an infectious agent associated with said infectiousdisease. In certain embodiments, the molecules of the invention aretoxic to the infectious agent, enhance immune response against saidagent or enhance effector function against said agent, relative to theimmune response in the absence of said molecule. Infectious diseasesthat can be treated or prevented by the molecules of the invention arecaused by infectious agents including but not limited to viruses,bacteria, fungi, protozae, and viruses.

Viral diseases that can be treated or prevented using the molecules ofthe invention in conjunction with the methods of the present inventioninclude, but are not limited to, those caused by hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral miningitis,encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by bacteria include, but are not limited to,mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borreliaburgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,streptococcus, staphylococcus, mycobacterium, tetanus, pertissus,cholera, plague, diptheria, chlamydia, S. aureus and legionella.

Protozoal diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by protozoa include, but are not limited to,leishmania, kokzidioa, trypanosoma or malaria.

Parasitic diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by parasites include, but are not limited to,chlamydia and rickettsia.

According to one aspect of the invention, molecules of the inventioncomprising at least one epitope binding domain specific for aninfectious agent exhibit an antibody effector function towards saidagent, e.g., a pathogenic protein. Examples of infectious agents includebut are not limited to bacteria (e.g., Escherichia coli, Klebsiellapneumoniae, Staphylococcus aureus, Enterococcus faecials, Candidaalbicans, Proteus vulgaris, Staphylococcus viridans, and Pseudomonasaeruginosa), a pathogen (e.g., B-lymphotropic papovavirus (LPV);Bordatella pertussis; Borna Disease virus (BDV); Bovine coronavirus;Choriomeningitis virus; Dengue virus; a virus, E. coli; Ebola; Echovirus1; Echovirus-11 (EV); Endotoxin (LPS); Enteric bacteria; Enteric Orphanvirus; Enteroviruses; Feline leukemia virus; Foot and mouth diseasevirus; Gibbon ape leukemia virus (GALV); Gram-negative bacteria;Heliobacter pylori; Hepatitis B virus (HBV); Herpes Simplex Virus;HIV-1; Human cytomegalovirus; Human coronovirus; Influenza A, B & C;Legionella; Leishmania mexicana; Listeria monocytogenes; Measles virus;Meningococcus; Morbilliviruses; Mouse hepatitis virus; Murine leukemiavirus; Murine gamma herpes virus; Murine retrovirus; Murine coronavirusmouse hepatitis virus; Mycobacterium avium-M; Neisseria gonorrhoeae;Newcastle disease virus; Parvovirus B19; Plasmodium falciparum; PoxVirus; Pseudomonas; Rotavirus; Samonella typhiurium; Shigella;Streptococci; T-cell lymphotropic virus 1; Vaccinia virus).

5.6.4 Detoxification

The invention also encompasses methods of detoxification in a subjectexposed to a toxin (e.g., a toxic drug molecule) comprisingadministering a therapeutically or prophylatically effective amount ofone or more molecules of the invention comprising at least one epitopebinding domain specific for the toxic drug molecule. In certainembodiments, binding of a molecule of the invention to the toxin reducesor eliminates the adverse physiological effect of said toxin. In yetother embodiments, binding of a diabody of the invention to the toxinincreases or enhances elimination, degradation or neutralization of thetoxin relative to elimination, degradation or neutralization in theabsence of said diabody. Immunotoxicotherapy in accordance with themethods of the invention can be used to treat overdoses or exposure todrugs including, but not limited to, digixin, PCP, cocaine, colchicine,and tricyclic antidepressants.

5.7 Combination Therapy

The invention further encompasses administering the molecules of theinvention in combination with other therapies known to those skilled inthe art for the treatment or prevention of cancer, autoimmune disease,infectious disease or intoxication, including but not limited to,current standard and experimental chemotherapies, hormonal therapies,biological therapies, immunotherapies, radiation therapies, or surgery.In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or more agents, therapeutic antibodies or other agentsknown to those skilled in the art for the treatment and/or prevention ofcancer, autoimmune disease, infectious disease or intoxication.

In certain embodiments, one or more molecule of the invention isadministered to a mammal, preferably a human, concurrently with one ormore other therapeutic agents useful for the treatment of cancer. Theterm “concurrently” is not limited to the administration of prophylacticor therapeutic agents at exactly the same time, but rather it is meantthat a molecule of the invention and the other agent are administered toa mammal in a sequence and within a time interval such that the moleculeof the invention can act together with the other agent to provide anincreased benefit than if they were administered otherwise. For example,each prophylactic or therapeutic agent (e.g., chemotherapy, radiationtherapy, hormonal therapy or biological therapy) may be administered atthe same time or sequentially in any order at different points in time;however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute. In various embodiments, the prophylactic or therapeutic agentsare administered less than 1 hour apart, at about 1 hour apart, at about1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart,at about 3 hours to about 4 hours apart, at about 4 hours to about 5hours apart, at about 5 hours to about 6 hours apart, at about 6 hoursto about 7 hours apart, at about 7 hours to about 8 hours apart, atabout 8 hours to about 9 hours apart, at about 9 hours to about 10 hoursapart, at about 10 hours to about 11 hours apart, at about 11 hours toabout 12 hours apart, no more than 24 hours apart or no more than 48hours apart. In preferred embodiments, two or more components areadministered within the same patient visit.

In other embodiments, the prophylactic or therapeutic agents areadministered at about 2 to 4 days apart, at about 4 to 6 days apart, atabout 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart. In preferred embodiments, the prophylactic or therapeutic agentsare administered in a time frame where both agents are still active. Oneskilled in the art would be able to determine such a time frame bydetermining the half life of the administered agents.

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a subject. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of a second agent and/or third agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

In certain embodiments, prophylactic or therapeutic agents areadministered in a cycle of less than about 3 weeks, about once every twoweeks, about once every 10 days or about once every week. One cycle cancomprise the administration of a therapeutic or prophylactic agent byinfusion over about 90 minutes every cycle, about 1 hour every cycle,about 45 minutes every cycle. Each cycle can comprise at least 1 week ofrest, at least 2 weeks of rest, at least 3 weeks of rest. The number ofcycles administered is from about 1 to about 12 cycles, more typicallyfrom about 2 to about 10 cycles, and more typically from about 2 toabout 8 cycles.

In yet other embodiments, the therapeutic and prophylactic agents of theinvention are administered in metronomic dosing regimens, either bycontinuous infusion or frequent administration without extended restperiods. Such metronomic administration can involve dosing at constantintervals without rest periods. Typically the therapeutic agents, inparticular cytotoxic agents, are used at lower doses. Such dosingregimens encompass the chronic daily administration of relatively lowdoses for extended periods of time. In preferred embodiments, the use oflower doses can minimize toxic side effects and eliminate rest periods.In certain embodiments, the therapeutic and prophylactic agents aredelivered by chronic low-dose or continuous infusion ranging from about24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3weeks to about 1 month to about 2 months, to about 3 months, to about 4months, to about 5 months, to about 6 months. The scheduling of suchdose regimens can be optimized by the skilled oncologist.

In other embodiments, courses of treatment are administered concurrentlyto a mammal, i.e., individual doses of the therapeutics are administeredseparately yet within a time interval such that molecules of theinvention can work together with the other agent or agents. For example,one component may be administered one time per week in combination withthe other components that may be administered one time every two weeksor one time every three weeks. In other words, the dosing regimens forthe therapeutics are carried out concurrently even if the therapeuticsare not administered simultaneously or within the same patient visit.

When used in combination with other prophylactic and/or therapeuticagents, the molecules of the invention and the prophylactic and/ortherapeutic agent can act additively or, more preferably,synergistically. In one embodiment, a molecule of the invention isadministered concurrently with one or more therapeutic agents in thesame pharmaceutical composition. In another embodiment, a molecule ofthe invention is administered concurrently with one or more othertherapeutic agents in separate pharmaceutical compositions. In stillanother embodiment, a molecule of the invention is administered prior toor subsequent to administration of another prophylactic or therapeuticagent. The invention contemplates administration of a molecule of theinvention in combination with other prophylactic or therapeutic agentsby the same or different routes of administration, e.g., oral andparenteral. In certain embodiments, when a molecule of the invention isadministered concurrently with another prophylactic or therapeutic agentthat potentially produces adverse side effects including, but notlimited to, toxicity, the prophylactic or therapeutic agent canadvantageously be administered at a dose that falls below the thresholdthat the adverse side effect is elicited.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

5.7.1 Anti-Cancer Agents

In a specific embodiment, the methods of the invention encompass theadministration of one or more molecules of the invention with one ormore therapeutic agents used for the treatment and/or prevention ofcancer. In one embodiment, angiogenesis inhibitors may be administeredin combination with the molecules of the invention. Angiogenesisinhibitors that can be used in the methods and compositions of theinvention include but are not limited to: Angiostatin (plasminogenfragment); antiangiogenic antithrombin III; Angiozyme; ABT-627; Bay12-9566; Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor(CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; CombretastatinA-4; Endostatin (collagen XVIII fragment); Fibronectin fragment;Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment;HMV833; Human chorionic gonadotropin (hCG); IM-862; Interferonalpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin-12;Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinaseinhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAbIMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placental ribonucleaseinhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4);Prinomastat; Prolactin 16 kDa fragment; Proliferin-related protein(PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU5416; SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate;thalidomide; Thrombospondin-1 (TSP-1); TNP-470; Transforming growthfactor-beta (TGF-b); Vasculostatin; Vasostatin (calreticulin fragment);ZD6126; ZD 6474; farnesyl transferase inhibitors (FTI); andbisphosphonates.

Anti-cancer agents that can be used in combination with the molecules ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-1 a; interferon gamma-1 b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to ZENAPAX® (daclizumab) (RochePharmaceuticals, Switzerland) which is an immunosuppressive, humanizedanti-CD25 monoclonal antibody for the prevention of acute renalallograft rejection; PANOREX™ which is a murine anti-17-IA cell surfaceantigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXINT™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody (IDECPharm/Mitsubishi); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 is a human anti-TGF-β₂ antibody (Cambridge Ab Tech).Other examples of therapeutic antibodies that can be used in accordancewith the invention are presented in Table 8.

TABLE 8 Anti-cancer therapeutic antibodies Company Product DiseaseTarget Abgenix ABX-EGF Cancer EGF receptor AltaRex OvaRex ovarian cancertumor antigen CA125 BravaRex metastatic tumor antigen cancers MUC1Antisoma Theragyn ovarian cancer PEM antigen (pemtumomabytrrium-90)Therex breast cancer PEM antigen Boehringer Blvatuzumab head & neck CD44Ingelheim cancer Centocor/J&J Panorex Colorectal 17-1A cancer ReoProPTCA gp IIIb/IIIa Corixa ReoPro Acute MI gp IIIb/IIIa ReoPro Ischemicstroke gp IIIb/IIIa Bexocar NHL CD20 CRC MAb, idiotypic 105AD7colorectal cancer gp72 Technology vaccine Crucell Anti-EpCAM cancerEp-CAM Cytoclonal MAb, lung cancer non-small cell NA lung cancerGenentech Herceptin metastatic breast HER-2 cancer Herceptin early stageHER-2 breast cancer Rituxan Relapsed/refractory CD20 low-grade orfollicular NHL Rituxan intermediate & CD20 high-grade NHL MAb-VEGFNSCLC, VEGF metastatic MAb-VEGF Colorectal VEGF cancer, metastatic AMDFab age-related CD18 macular degeneration E-26 (2^(nd) gen. IgE)allergic asthma IgE & rhinitis IDEC Zevalin (Rituxan + yttrium-90) lowgrade of CD20 follicular, relapsed or refractory, CD20-positive, B-cellNHL and Rituximab- refractory NHL ImClone Cetuximab + innotecanrefractory EGF receptor colorectal carcinoma Cetuximab + cisplatin &newly diagnosed EGF receptor radiation or recurrent head & neck cancerCetuximab + newly diagnosed EGF receptor gemcitabine metastaticpancreatic carcinoma Cetuximab + cisplatin + recurrent or EGF receptor5FU or Taxol metastatic head & neck cancer Cetuximab + newly diagnosedEGF receptor carboplatin + paclitaxel non-small cell lung carcinomaCetuximab + cisplatin head & neck EGF receptor cancer (extensiveincurable local-regional disease & distant metasteses) Cetuximab +radiation locally advanced EGF receptor head & neck carcinoma BEC2 +Bacillus small cell lung mimics Calmette Guerin carcinoma gangliosideGD3 BEC2 + Bacillus melanoma mimics Calmette Guerin ganglioside GD3IMC-1C11 colorectal cancer VEGF-receptor with liver metasteses ImmonoGennuC242-DM1 Colorectal, nuC242 gastric, and pancreatic cancerImmunoMedics LymphoCide Non-Hodgkins CD22 lymphoma LymphoCide Y-90Non-Hodgkins CD22 lymphoma CEA-Cide metastatic solid CEA tumors CEA-CideY-90 metastatic solid CEA tumors CEA-Scan (Tc-99m- colorectal cancer CEAlabeled arcitumomab) (radioimaging) CEA-Scan (Tc-99m- Breast cancer CEAlabeled arcitumomab) (radioimaging) CEA-Scan (Tc-99m- lung cancer CEAlabeled arcitumomab) (radioimaging) CEA-Scan (Tc-99m- intraoperative CEAlabeled arcitumomab) tumors (radio imaging) LeukoScan (Tc-99m- softtissue CEA labeled sulesomab) infection (radioimaging) LymphoScan(Tc-99m- lymphomas CD22 labeled) (radioimaging) AFP-Scan (Tc-99m- liver7 gem-cell AFP labeled) cancers (radioimaging) Intracel HumaRAD-HN head& neck NA (+yttrium-90) cancer HumaSPECT colorectal NA imaging MedarexMDX-101 (CTLA-4) Prostate and CTLA-4 other cancers MDX-210 (her-2Prostate cancer HER-2 overexpression) MDX-210/MAK Cancer HER-2 MedImmuneVitaxin Cancer αvβ₃ Merck KGaA MAb 425 Various cancers EGF receptorIS-IL-2 Various cancers Ep-CAM Millennium Campath chronic CD52(alemtuzumab) lymphocytic leukemia NeoRx CD20-streptavidin Non-HodgkinsCD20 (+biotin-yttrium 90) lymphoma Avidicin (albumin + metastatic NANRLU13) cancer Peregrine Oncolym (+iodine-131) Non-Hodgkins HLA-DR 10lymphoma beta Cotara (+iodine-131) unresectable DNA-associated malignantproteins glioma Pharmacia C215 (+staphylococcal pancreatic NACorporation enterotoxin) cancer MAb, lung/kidney lung & kidney NA cancercancer nacolomab tafenatox colon & NA (C242 + staphylococcal pancreaticenterotoxin) cancer Protein Design Nuvion T cell CD3 Labs malignanciesSMART M195 AML CD33 SMART 1D10 NHL HLA-DR antigen Titan CEAVaccolorectal CEA cancer, advanced TriGem metastatic GD2- melanoma &ganglioside small cell lung cancer TriAb metastatic breast MUC-1 cancerTrilex CEAVac colorectal CEA cancer, advanced TriGem metastatic GD2-melanoma & ganglioside small cell lung cancer TriAb metastatic breastMUC-1 cancer Viventia NovoMAb-G2 Non-Hodgkins NA Biotech radiolabeledlymphoma Monopharm C colorectal & SK-1 antigen pancreatic carcinomaGlioMAb-H (+gelonin gliorna, NA toxin) melanoma & neuroblastoma XomaRituxan Relapsed/refractory CD20 low-grade or follicular NHL Rituxanintermediate & CD20 high-grade NHL ING-1 adenomcarcinoma Ep-CAM

5.7.2 Immunomodulatory Agents and Anti-Inflammatory Agents

The present invention provides methods of treatment for autoimmunediseases and inflammatory diseases comprising administration of themolecules of the invention in conjunction with other treatment agents.Examples of immunomodulatory agents include, but are not limited to,methothrexate, ENBREL, REMICADE™, leflunomide, cyclophosphamide,cyclosporine A, and macrolide antibiotics (e.g., FK506 (tacrolimus)),methylprednisolone (MP), corticosteroids, steroids, mycophenolatemofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,and cytokine receptor modulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINET™), fenoprofen (NALFON™), indomethacin (INDOCINT™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTINT™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

A non-limiting example of the antibodies that can be used for thetreatment or prevention of inflammatory disorders in conjunction withthe molecules of the invention is presented in Table 9, and anon-limiting example of the antibodies that can used for the treatmentor prevention of autoimmune disorder is presented in Table 10.

TABLE 9 Therapeutic antibodies for the treatment of inflammatorydiseases Antibody Target Product Name Antigen Type Isotype SponsorsIndication 5G1.1 Complement Humanized IgG Alexion Rheumatoid (C5) PharmInc Arthritis 5G1.1 Complement Humanized IgG Alexion SLE (C5) Pharm Inc5G1.1 Complement Humanized IgG Alexion Nephritis (C5) Pharm Inc 5G1.1-SCComplement Humanized ScFv Alexion Cardiopulmonary (C5) Pharm Inc Bypass5G1.1-SC Complement Humanized ScFv Alexion Myocardial (C5) Pharm IncInfarction 5G1.1-SC Complement Humanized ScFv Alexion Angioplasty (C5)Pharm Inc ABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgGAbgenix Inc Allograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix IncPsoriasis Antegren VLA-4 Humanized IgG Athena/Elan Multiple SclerosisAnti-CD11a CD11a Humanized IgG1 Genentech Psoriasis Inc/Xoma Anti-CD18CD18 Humanized Fab′2 Genentech Inc Myocardial infarction Anti-LFA1 CD18Murine Fab′2 Pasteur- Allograft Merieux/ rejection Immunotech AntovaCD40L Humanized IgG Biogen Allograft rejection Antova CD40L HumanizedIgG Biogen SLE BTI-322 CD2 Rat IgG Medimmune GvHD, Inc Psoriasis CDP571TNF-alpha Humanized IgG4 Celltech Crohn's CDP571 TNF-alpha HumanizedIgG4 Celltech Rheumatoid Arthritis CDP850 E-selectin Humanized CelltechPsoriasis Corsevin M Fact VII Chimeric Centocor Anticoagulant D2E7TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18 HumanizedICOS Pharm Multiple Inc Sclerosis Hu23F2G CD11/18 Humanized IgG ICOSPharm Stroke Inc IC14 CD14 ICOS Pharm Toxic shock Inc ICM3 ICAM-3Humanized ICOS Pharm Psoriasis Inc IDEC-114 CD80 Primatised IDEC Pharm/Psoriasis Mitsubishi IDEC-131 CD40L Humanized IDEC SLE Pharm/EisaiIDEC-131 CD40L Humanized IDEC Multiple Pharm/Eisai Sclerosis IDEC-151CD4 Primatised IgG1 IDEC Rheumatoid Pharm/Glaxo Arthritis SmithKlineIDEC-152 CD23 Primatised IDEC Pharm Asthma/ Allergy Infliximab TNF-alphaChimeric IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Centocor Crohn's LDP-01 beta2- Humanized IgG MillenniumInc Stroke integrin (LeukoSite Inc.) LDP-01 beta2- Humanized IgGMillennium Inc Allograft integrin (LeukoSite Inc.) rejection LDP-02alpha4beta7 Humanized Millennium Inc Ulcerative (LeukoSite Inc.) ColitisMAK-195F TNF alpha Murine Fab′2 Knoll Pharm, Toxic shock BASF MDX-33CD64 (FcR) Human Medarex/Centeon Autoimmune haematogical disordersMDX-CD4 CD4 Human IgG Medarex/Eisai/ Rheumatoid Genmab ArthritisMEDI-507 CD2 Humanized Medimmune Psoriasis Inc MEDI-507 CD2 HumanizedMedimmune GvHD Inc OKT4A CD4 Humanized IgG Ortho Biotech Allograftrejection OrthoClone CD4 Humanized IgG Ortho Biotech Autoimmune OKT4Adisease Orthoclone/ CD3 Murine mIgG2a Ortho Biotech Allograft anti-CD3rejection OKT3 RepPro/ gpIIbIIIa Chimeric Fab Centocor/LillyComplications Abciximab of coronary angioplasty rhuMab-E25 IgE HumanizedIgG1 Genentech/ Asthma/ Novartis/Tanox Allergy Biosystems SB-240563 IL5Humanized GlaxoSmithKline Asthma/ Allergy SB-240683 IL-4 HumanizedGlaxoSmithKline Asthma/ Allergy SCH55700 IL-5 Humanized Celltech/Asthma/ Schering Allergy Simulect CD25 Chimeric IgG1 Novartis AllograftPharm rejection SMART CD3 Humanized Protein Autoimmune a-CD3 Design Labdisease SMART CD3 Humanized Protein Allograft a-CD3 Design Lab rejectionSMART CD3 Humanized IgG Protein Psoriasis a-CD3 Design Lab Zenapax CD25Humanized IgG1 Protein Allograft Design rejection Lab/Hoffman- La Roche

TABLE 10 Therapeutic antibodies for the treatment of autoimmunedisorders Antibody Indication Target Antigen ABX-RB2 antibody to CBLantigen on T cells, B cells and NK cells fully human antibody from theXenomouse 5c8 (Anti CD-40 an Phase II trials were CD-40 antigenantibody) halted in October, 1999 examine “adverse events” IDEC 131systemic lupus anti CD40 erythyematous (SLE) humanized IDEC 151rheumatoid arthritis primatized; anti-CD4 IDEC 152 Asthma primatized;anti-CD23 IDEC 114 Psoriasis primatized anti-CD80 MEDI-507 rheumatoidarthritis; anti-CD2 multiple sclerosis Crohn's disease Psoriasis LDP-02(anti-b7 inflammatory bowel a4b7 integrin receptor on white mAb) diseaseblood cells (leukocytes) Chron's disease ulcerative colitis SMART Anti-autoimmune disorders Anti-Gamma Interferon Gamma Interferon antibodyVerteportin rheumatoid arthritis MDX-33 blood disorders causedmonoclonal antibody against by autoimmune reactions FcRI receptorsIdiopathic Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemiaMDX-CD4 treat rheumatoid arthritis monoclonal antibody against CD4 andother autoimmunity receptor molecule VX-497 autoimmune disordersinhibitor of inosine multiple sclerosis monophosphate dehydrogenaserheumatoid arthritis (enzyme needed to make new inflammatory bowel RNAand DNA disease used in production of nucleotides lupus needed forlymphocyte psoriasis proliferation) VX-740 rheumatoid arthritisinhibitor of ICE interleukin-1 beta (converting enzyme controls pathwaysleading to aggressive immune response) VX-745 specific to inflammationinhibitor of P38MAP kinase involved in chemical mitogen activatedprotein kinase signalling of immune response onset and progression ofinflammation Enbrel (etanercept) targets TNF (tumor necrosis factor)IL-8 fully human monoclonal antibody against IL-8 (interleukin 8) ApogenMP4 recombinant antigen selectively destroys disease associated T-cellsinduces apoptosis T-cells eliminated by programmed cell death no longerattack body's own cells specific apogens target specific T-cells

5.7.3 Agents for Use in the Treatment of Infectious Disease

In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or additional therapeutic agents known to those skilled inthe art for the treatment and/or prevention of an infectious disease.The invention contemplates the use of the molecules of the invention incombination with antibiotics known to those skilled in the art for thetreatment and or prevention of an infectious disease. Antibiotics thatcan be used in combination with the molecules of the invention include,but are not limited to, macrolide (e.g., tobramycin (Tobi®)), acephalosporin (e.g., cephalexin (Keflex®), cephradine (Velosef®),cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime(Suprax®) or cefadroxil (Duricef®)), a clarithromycin (e.g.,clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin(EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen VeeK®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®)or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g., apramycin,arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin,undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, andspectinomycin), amphenicol antibiotics (e.g., azidamfenicol,chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics(e.g., rifamide and rifampin), carbacephems (e.g., loracarbef),carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g.,cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran,cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefinetazole, and cefminox), monobactams (e.g.,aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, andmoxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil,amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillinsodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamatehydriodide, penicillin o-benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin clinafloxacin,flumequine, and grepagloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

In certain embodiments, the molecules of the invention can beadministered in combination with a therapeutically or prophylacticallyeffective amount of one or more antifungal agents. Antifungal agentsthat can be used in combination with the molecules of the inventioninclude but are not limited to amphotericin B, itraconazole,ketoconazole, fluconazole, intrathecal, flucytosine, miconazole,butoconazole, clotrimazole, nystatin, terconazole, tioconazole,ciclopirox, econazole, haloprogrin, naftifine, terbinafine,undecylenate, and griseofuldin.

In some embodiments, the molecules of the invention can be administeredin combination with a therapeutically or prophylactically effectiveamount of one or more anti-viral agent. Useful anti-viral agents thatcan be used in combination with the molecules of the invention include,but are not limited to, protease inhibitors, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors and nucleoside analogs. Examples of antiviral agents includebut are not limited to zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin, as well as foscarnet,amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir,ritonavir, the alpha-interferons; adefovir, clevadine, entecavir,pleconaril.

5.8 Vaccine Therapy

The invention further encompasses using a composition of the inventionto induce an immune response against an antigenic or immunogenic agent,including but not limited to cancer antigens and infectious diseaseantigens (examples of which are disclosed infra). The vaccinecompositions of the invention comprise one or more antigenic orimmunogenic agents to which an immune response is desired, wherein theone or more antigenic or immunogenic agents is coated with a variantantibody of the invention that has an enhanced affinity to FcγRIIIA. Thevaccine compositions of the invention are particularly effective ineliciting an immune response, preferably a protective immune responseagainst the antigenic or immunogenic agent.

In some embodiments, the antigenic or immunogenic agent in the vaccinecompositions of the invention comprises a virus against which an immuneresponse is desired. The viruses may be recombinant or chimeric, and arepreferably attenuated. Production of recombinant, chimeric, andattenuated viruses may be performed using standard methods known to oneskilled in the art. The invention encompasses a live recombinant viralvaccine or an inactivated recombinant viral vaccine to be formulated inaccordance with the invention. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In a specific embodiment, the recombinant virus is non-pathogenic to thesubject to which it is administered. In this regard, the use ofgenetically engineered viruses for vaccine purposes may require thepresence of attenuation characteristics in these strains. Theintroduction of appropriate mutations (e.g., deletions) into thetemplates used for transfection may provide the novel viruses withattenuation characteristics. For example, specific missense mutationswhich are associated with temperature sensitivity or cold adaptation canbe made into deletion mutations. These mutations should be more stablethan the point mutations associated with cold or temperature sensitivemutants and reversion frequencies should be extremely low. RecombinantDNA technologies for engineering recombinant viruses are known in theart and encompassed in the invention. For example, techniques formodifying negative strand RNA viruses are known in the art, see, e.g.,U.S. Pat. No. 5,166,057, which is incorporated herein by reference inits entirety.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed for use in the intradermal vaccine formulations of theinvention. Such viruses would go through only one or a few rounds ofreplication within the host. When used as a vaccine, the recombinantvirus would go through limited replication cycle(s) and induce asufficient level of immune response but it would not go further in thehuman host and cause disease. Alternatively, inactivated (killed) virusmay be formulated in accordance with the invention. Inactivated vaccineformulations may be prepared using conventional techniques to “kill” thechimeric viruses. Inactivated vaccines are “dead” in the sense thattheir infectivity has been destroyed. Ideally, the infectivity of thevirus is destroyed without affecting its immunogenicity. In order toprepare inactivated vaccines, the chimeric virus may be grown in cellculture or in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled.

In certain embodiments, completely foreign epitopes, including antigensderived from other viral or non-viral pathogens can be engineered intothe virus for use in the intradermal vaccine formulations of theinvention. For example, antigens of non-related viruses such as HIV(gp160, gp120, gp41) parasite antigens (e.g., malaria), bacterial orfungal antigens or tumor antigens can be engineered into the attenuatedstrain.

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in the intradermal vaccineformulations. Preferably, heterologous gene sequences are moieties andpeptides that act as biological response modifiers. Preferably, epitopesthat induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention include, but are not limited to, influenza andparainfluenza hemagglutinin neuraminidase and fusion glycoproteins suchas the HN and F genes of human PIV3. In yet another embodiment,heterologous gene sequences that can be engineered into the chimericviruses include those that encode proteins with immuno-modulatingactivities. Examples of immuno-modulating proteins include, but are notlimited to, cytokines, interferon type 1, gamma interferon, colonystimulating factors, interleukin-1, -2, -4, -5, -6, -12, and antagonistsof these agents.

In yet other embodiments, the invention encompasses pathogenic cells orviruses, preferably attenuated viruses, which express the variantantibody on their surface.

In alternative embodiments, the vaccine compositions of the inventioncomprise a fusion polypeptide wherein an antigenic or immunogenic agentis operatively linked to a variant antibody of the invention that has anenhanced affinity for FcγRIIIA. Engineering fusion polypeptides for usein the vaccine compositions of the invention is performed using routinerecombinant DNA technology methods and is within the level of ordinaryskill.

The invention further encompasses methods to induce tolerance in asubject by administering a composition of the invention. Preferably acomposition suitable for inducing tolerance in a subject comprises anantigenic or immunogenic agent coated with a variant antibody of theinvention, wherein the variant antibody has a higher affinity toFcγRIIB. Although not intending to be bound by a particular mechanism ofaction, such compositions are effective in inducing tolerance byactivating the FcγRIIB meditated inhibitory pathway.

5.9 compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising molecules of the invention (i.e., diabodies) comprisingmultiple epitope binding domains and, optionally, an Fc domain (orportion thereof). The invention also provides methods of treatment,prophylaxis, and amelioration of one or more symptoms associated with adisease, disorder or infection by administering to a subject aneffective amount of a fusion protein or a conjugated molecule of theinvention, or a pharmaceutical composition comprising a fusion proteinor a conjugated molecule of the invention. In a preferred aspect, anantibody, a fusion protein, or a conjugated molecule, is substantiallypurified (i.e., substantially free from substances that limit its effector produce undesired side-effects). In a specific embodiment, thesubject is an animal, preferably a mammal such as non-primate (e.g.,cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkeysuch as, a cynomolgous monkey and a human). In a preferred embodiment,the subject is a human. In yet another preferred embodiment, theantibody of the invention is from the same species as the subject.

Various delivery systems are known and can be used to administer acomposition comprising molecules of the invention, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the antibody or fusion protein, receptor-mediatedendocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In VitroGene Transformation By A Soluble DNA Carrier System,” J. Biol. Chem.262:4429-4432), construction of a nucleic acid as part of a retroviralor other vector, etc. Methods of administering a molecule of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).In a specific embodiment, the molecules of the invention areadministered intramuscularly, intravenously, or subcutaneously. Thecompositions may be administered by any convenient route, for example,by infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety.

The invention also provides that the molecules of the invention arepackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of antibody. In one embodiment, themolecules of the invention are supplied as a dry sterilized lyophilizedpowder or water free concentrate in a hermetically sealed container andcan be reconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, the moleculesof the invention are supplied as a dry sterile lyophilized powder in ahermetically sealed container at a unit dosage of at least 5 mg, morepreferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilizedmolecules of the invention should be stored at between 2 and 8° C. intheir original container and the molecules should be administered within12 hours, preferably within 6 hours, within 5 hours, within 3 hours, orwithin 1 hour after being reconstituted. In an alternative embodiment,molecules of the invention are supplied in liquid form in a hermeticallysealed container indicating the quantity and concentration of themolecule, fusion protein, or conjugated molecule. Preferably, the liquidform of the molecules of the invention are supplied in a hermeticallysealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml,at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, atleast 150 mg/ml, at least 200 mg/ml of the molecules.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For diabodies encompassed by the invention, the dosage administered to apatient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. The dosage andfrequency of administration of diabodies of the invention may be reducedor altered by enhancing uptake and tissue penetration of the diabodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the molecules of the inventionadministered to a patient may be from 0.01 mg to 1000 mg/day when usedas single agent therapy. In another embodiment the molecules of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidmolecules are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a molecule of the invention, care must be taken touse materials to which the molecule does not absorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer (1990) “New Methods Of DrugDelivery,” Science 249:1527-1533); Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more molecules of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al. (1996) “IntratumoralRadioimmunotheraphy Of A Human Colon Cancer Xenograft Using ASustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.(1995) “Antibody Mediated Lung Targeting Of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody ForCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation OfRecombinant Humanized Monoclonal Antibody For Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton, (1987) “Implantable Pumps,” CRC Crit. Rev. Biomed. Eng.14:201-240; Buchwald et al. (1980) “Long-Term, Continuous IntravenousHeparin Administration By An Implantable Infusion Pump In AmbulatoryPatients With Recurrent Venous Thrombosis,” Surgery 88:507-516; andSaudek et al. (1989) “A Preliminary Trial Of The ProgrammableImplantable Medication System For Insulin Delivery,” N. Engl. J. Med.321:574-579). In another embodiment, polymeric materials can be used toachieve controlled release of antibodies (see e.g., Medical Applicationsof Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,Fla. (1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Levy et al.(1985) “Inhibition Of Calcification Of Bioprosthetic Heart Valves ByLocal Controlled-Release Diphosphonate,” Science 228:190-192; During etal. (1989) “Controlled Release Of Dopamine From A Polymeric BrainImplant: In Vivo Characterization,” Ann. Neurol. 25:351-356; Howard etal. (1989) “Intracerebral Drug Delivery In Rats With Lesion-InducedMemory Deficits,” J. Neurosurg. 7(1):105-112); U.S. Pat. No. 5,679,377;U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No.5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; andPCT Publication No. WO 99/20253). Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe therapeutic target (e.g., the lungs), thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). In anotherembodiment, polymeric compositions useful as controlled release implantsare used according to Dunn et al. (See U.S. Pat. No. 5,945,155). Thisparticular method is based upon the therapeutic effect of the in situcontrolled release of the bioactive material from the polymer system.The implantation can generally occur anywhere within the body of thepatient in need of therapeutic treatment. In another embodiment, anon-polymeric sustained delivery system is used, whereby a non-polymericimplant in the body of the subject is used as a drug delivery system.Upon implantation in the body, the organic solvent of the implant willdissipate, disperse, or leach from the composition into surroundingtissue fluid, and the non-polymeric material will gradually coagulate orprecipitate to form a solid, microporous matrix (See U.S. Pat. No.5,888,533).

Controlled release systems are discussed in the review by Langer (1990,“New Methods Of Drug Delivery,” Science 249:1527-1533). Any techniqueknown to one of skill in the art can be used to produce sustainedrelease formulations comprising one or more therapeutic agents of theinvention. See, e.g., U.S. Pat. No. 4,526,938; International PublicationNos. WO 91/05548 and WO 96/20698; Ning et al. (1996) “IntratumoralRadioimmunotheraphy Of A Human Colon Cancer Xenograft Using ASustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.(1995) “Antibody Mediated Lung Targeting Of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody ForCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation OfRecombinant Humanized Monoclonal Antibody For Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding a diabody of the invention, the nucleic acid canbe administered in vivo to promote expression of its encoded diabody, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al. (1991) “Antennapedia Homeobox Peptide Regulates NeuralMorphogenesis,” Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of molecules of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with molecules of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the molecules used for treatment may increase or decrease overthe course of a particular treatment.

5.9.1 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of one or more molecules of theinvention and a pharmaceutically acceptable carrier.

The invention also encompasses pharmaceutical compositions comprising adiabody molecule of the invention and a therapeutic antibody (e.g.,tumor specific monoclonal antibody) that is specific for a particularcancer antigen, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

5.9.2 Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingmolecules of the invention are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel et al.(1993) “Human Gene Therapy,” Clinical Pharmacy 12:488-505; Wu et al.(1991) “Delivery Systems For Gene Therapy,” Biotherapy 3:87-95;Tolstoshev (1993) “Gene Therapy, Concepts, Current Trials And FutureDirections,” Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993)“The Basic Science Of Gene Therapy,” Science 260:926-932; and Morgan etal. (1993) “Human Gene Therapy,” Ann. Rev. Biochem. 62:191-217. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding a diabody of the invention, said nucleic acids being partof an expression vector that expresses the antibody in a suitable host.In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the antibody coding region,said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the antibody coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the antibody encoding nucleic acids(Koller et al. (1989) “Inactivating The Beta 2-Microglobulin Locus InMouse Embryonic Stem Cells By Homologous Recombination,” Proc. Natl.Acad. Sci. USA 86:8932-8935; and Zijlstra et al. (1989) “Germ-LineTransmission Of A Disrupted Beta 2-Microglobulin Gene Produced ByHomologous Recombination In Embryonic Stem Cells,” Nature 342:435-438).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expresses the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a an antigen subject to receptor-mediatedendocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In VitroGene Transformation By A Soluble DNA Carrier System,” J. Biol. Chem.262:4429-4432) (which can be used to target cell types specificallyexpressing the receptors), etc. In another embodiment, nucleicacid-antigen complexes can be formed in which the antigen comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (See, e.g., PCT Publications WO 92/06180;WO 92/22635; WO92/20316; WO93/14188; WO 93/20221). Alternatively, thenucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression, by homologous recombination (Koller et al.(1989) “Inactivating The Beta 2-Microglobulin Locus In Mouse EmbryonicStem Cells By Homologous Recombination,” Proc. Natl. Acad. Sci. USA86:8932-8935; and Zijlstra et al. (1989) “Germ-Line Transmission Of ADisrupted Beta 2-Microglobulin Gene Produced By Homologous RecombinationIn Embryonic Stem Cells,” Nature 342:435-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding a molecule of the invention (e.g., a diabody or afusion protein) are used. For example, a retroviral vector can be used(See Miller et al. (1993) “Use Of Retroviral Vectors For Gene TransferAnd Expression,” Meth. Enzymol. 217:581-599). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding the antibody or a fusion protein to be used in genetherapy are cloned into one or more vectors, which facilitate deliveryof the nucleotide sequence into a subject. More detail about retroviralvectors can be found in Boesen et al. (1993) “Circumvention OfChemotherapy-Induced Myelosuppression By Transfer Of The Mdr 1 Gene,”Biotherapy 6:291-302), which describes the use of a retroviral vector todeliver the mdr 1 gene to hematopoietic stem cells in order to make thestem cells more resistant to chemotherapy. Other references illustratingthe use of retroviral vectors in gene therapy are: Clowes et al. (1994)“Long-Term Biological Response Of Injured Rat Carotid Artery Seeded WithSmooth Muscle Cells Expressing Retrovirally Introduced Human Genes,” J.Clin. Invest. 93:644-651; Keim et al. (1994) “Retrovirus-Mediated GeneTransduction Into Canine Peripheral Blood Repopulating Cells,” Blood83:1467-1473; Salmons et al. (1993) “Targeting Of Retroviral Vectors ForGene Therapy,” Human Gene Therapy 4:129-141; and Grossman et al. (1993)“Retroviruses: Delivery Vehicle To The Liver,” Curr. Opin. Genetics andDevel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky et al. (1993, “GeneTherapy: Adenovirus Vectors,” Current Opinion in Genetics andDevelopment 3:499-503) present a review of adenovirus-based genetherapy. Bout et al. (1994, “Lung Gene Therapy: In VivoAdenovirus-Mediated Gene Transfer To Rhesus Monkey Airway Epithelium,”Human Gene Therapy, 5:3-10) demonstrated the use of adenovirus vectorsto transfer genes to the respiratory epithelia of rhesus monkeys. Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld et al. (1991) “Adenovirus-Mediated Transfer Of A RecombinantAlpha 1-Antitrypsin Gene To The Lung Epithelium In Vivo,” Science252:431-434; Rosenfeld et al. (1992) “In Vivo Transfer Of The HumanCystic Fibrosis Transmembrane Conductance Regulator Gene To The AirwayEpithelium,” Cell 68:143-155; Mastrangeli et al. (1993) “Diversity OfAirway Epithelial Cell Targets For In Vivo RecombinantAdenovirus-Mediated Gene Transfer,” J. Clin. Invest. 91:225-234; PCTPublication WO94/12649; and Wang et al. (1995) “A Packaging Cell LineFor Propagation Of Recombinant Adenovirus Vectors Containing Two LethalGene-Region Deletions,” Gene Therapy 2:775-783. In a preferredembodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g., Walsh et al. (1993) “Gene Therapy For HumanHemoglobinopathies,” Proc. Soc. Exp. Biol. Med. 204:289-300 and U.S.Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to, transfection, electroporation,microinjection, infection with a viral or bacteriophage vector,containing the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (See, e.g., Loeffler et al. (1993) “GeneTransfer Into Primary And Established Mammalian Cell Lines WithLipopolyamine-Coated DNA,” Meth. Enzymol. 217:599-618, Cotten et al.(1993) “Receptor-Mediated Transport Of DNA Into Eukaryotic Cells,” Meth.Enzymol. 217:618-644) and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (See e.g., PCT Publication WO94/08598; Stemple et al. (1992) “Isolation Of A Stem Cell For NeuronsAnd Glia From The Mammalian Neural Crest,” Cell 7 1:973-985; Rheinwald(1980) “Serial Cultivation Of Normal Human Epidermal Keratinocytes,”Meth. Cell Bio. 21A:229-254; and Pittelkow et al. (1986) “New TechniquesFor The In Vitro Culture Of Human Skin Keratinocytes And Perspectives OnTheir Use For Grafting Of Patients With Extensive Burns,” Mayo ClinicProc. 61:771-777).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

5.9.3 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with the molecules of the invention.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a disease can also be included in thepharmaceutical pack or kit. The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more molecules of theinvention. In another embodiment, a kit further comprises one or moreother prophylactic or therapeutic agents useful for the treatment ofcancer, in one or more containers. In another embodiment, a kit furthercomprises one or more cytotoxic antibodies that bind one or more cancerantigens associated with cancer. In certain embodiments, the otherprophylactic or therapeutic agent is a chemotherapeutic. In otherembodiments, the prophylactic or therapeutic agent is a biological orhormonal therapeutic.

5.10 Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions, prophylactic, ortherapeutic agents of the invention are preferably tested in vitro, in acell culture system, and in an animal model organism, such as a rodentanimal model system, for the desired therapeutic activity prior to usein humans. For example, assays which can be used to determine whetheradministration of a specific pharmaceutical composition is desired,include cell culture assays in which a patient tissue sample is grown inculture, and exposed to or otherwise contacted with a pharmaceuticalcomposition of the invention, and the effect of such composition uponthe tissue sample is observed. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. In various specific embodiments, in vitroassays can be carried out with representative cells of cell typesinvolved in an autoimmune or inflammatory disorder (e.g., T cells), todetermine if a pharmaceutical composition of the invention has a desiredeffect upon such cell types.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary. Said aspectsinclude the temporal regime of administering the prophylactic and/ortherapeutic agents, and whether such agents are administered separatelyor as an admixture.

Preferred animal models for use in the methods of the invention are, forexample, transgenic mice expressing human FcγR5 on mouse effector cells,e.g., any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety) can be used in thepresent invention. Transgenic mice for use in the methods of theinvention include, but are not limited to, mice carrying human FcγRIIIA;mice carrying human FcγRIIA; mice carrying human FcγRIIB and humanFcγRIIIA; mice carrying human FcγRIIB and human FcγRIIA. Preferably,mutations showing the highest levels of activity in the functionalassays described above will be tested for use in animal model studiesprior to use in humans. Sufficient quantities of antibodies may beprepared for use in animal models using methods described supra, forexample using mammalian expression systems and purification methodsdisclosed and exemplified herein.

Mouse xenograft models may be used for examining efficacy of mouseantibodies generated against a tumor specific target based on theaffinity and specificity of the epitope bing domains of the diabodymolecule of the invention and the ability of the diabody to elicit animmune response (Wu et al. (2001) “Mouse Models For MultistepTumorigenesis,” Trends Cell Biol. 11: S2-9). Transgenic mice expressinghuman FcγRs on mouse effector cells are unique and are tailor-madeanimal models to test the efficacy of human Fc-FcγR interactions. Pairsof FcγRIIIA, FcγRIIIB and FcγRIIA transgenic mouse lines generated inthe lab of Dr. Jeffrey Ravetch (Through a licensing agreement withRockefeller U. and Sloan Kettering Cancer center) can be used such asthose listed in the Table 11 below.

TABLE 11 Mice Strains Strain Background Human FcR Nude/CD16A KO NoneNude/CD16A KO FcγRIIIA Nude/CD16A KO FcγR IIA Nude/CD16A KO FcγR IIA andIIIA Nude/CD32B KO None Nude/CD32B KO FcγR IIB

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneousanimal models of inflammatory arthritis and autoimmune rheumaticdiseases can also be used to assess the anti-inflammatory activity ofthe combination therapies of invention. The following are some assaysprovided as examples, and not by limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model aredescribed in Hansra P. et al. (2000) “Carrageenan-Induced Arthritis InThe Rat,” Inflammation, 24(2): 141-155. Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al. (1962) “Carrageenan-Induced Edema In Hind Paw Of TheRat As An Assay For Anti-Inflammatory Drugs” Proc. Soc. Exp. Biol Med.111, 544-547. This assay has been used as a primary in vivo screen forthe anti-inflammatory activity of most NSAIDs, and is consideredpredictive of human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

Additionally, animal models for inflammatory bowel disease can also beused to assess the efficacy of the combination therapies of invention(Kim et al. (1992) “Experimental Colitis In Animal Models,” Scand. J.Gastroentrol. 27:529-537; Strober (1985) “Animal Models Of InflammatoryBowel Disease—An Overview,” Dig. Dis. Sci. 30(12 Suppl):3S-10S).Ulcerative cholitis and Crohn's disease are human inflammatory boweldiseases that can be induced in animals. Sulfated polysaccharidesincluding, but not limited to amylopectin, carrageen, amylopectinsulfate, and dextran sulfate or chemical irritants including but notlimited to trinitrobenzenesulphonic acid (TNBS) and acetic acid can beadministered to animals orally to induce inflammatory bowel diseases.

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have been developed(Flanders et al. (1999) “Prevention Of Type 1 Diabetes From LaboratoryTo Public Health,” Autoimmunity 29:235-246; Rasmussen et al. (1999)“Models To Study The Pathogenesis Of Thyroid Autoimmunity,” Biochimie81:511-515; Foster (1999) “Relevance Of Systemic Lupus ErythematosusNephritis Animal Models To Human Disease,” Semin. Nephrol. 19:12-24).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

Preferred animal models for determining the therapeutic efficacy of themolecules of the invention are mouse xenograft models. Tumor cell linesthat can be used as a source for xenograft tumors include but are notlimited to, SKBR3 and MCF₇ cells, which can be derived from patientswith breast adenocarcinoma. These cells have both erbB2 and prolactinreceptors. SKBR3 cells have been used routinely in the art as ADCC andxenograft tumor models. Alternatively, OVCAR3 cells derived from a humanovarian adenocarcinoma can be used as a source for xenograft tumors.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

6. EXAMPLES 6.1 Design and Characterization of Covalent BispecificDiabodies

A monospecific covalent diabody and a bispecific covalent diabody wereconstructed to assess the recombinant production, purification andbinding characteristics of each. The affinity purified diabody moleculesthat were produced by the recombinant expression systems describedherein were found by SDS-PAGE and SEC analysis to consist of a singledimeric species. ELISA and SPR analysis further revealed that thecovalent bispecific diabody exhibited affinity for both target antigensand could bind both antigens simultaneously.

Materials and Methods:

Construction and Design of Polypeptide Molecules: Nucleic acidexpression vectors were designed to produce four polypeptide constructs,schematically represented in FIG. 2. Construct 1 (SEQ ID NO: 9)comprised the VL domain of humanized 2B6 antibody, which recognizesFcγRIIB, and the VH domain of humanized 3G8 antibody, which recognizesFcγR^(I)IIA. Construct 2 (SEQ ID NO: 11) comprised the VL domain ofHu3G8 and the VH domain of Hu2B6. Construct 3 (SEQ ID NO: 12) comprisedthe VL domain of Hu3G8 and the VH domain of Hu3G8. Construct 4 (SEQ IDNO: 13) comprised the VL domain of Hu2B6 and the VH domain of Hu2B6.

PCR and Expression Vector Construction: The coding sequences of the VLor VH domains were amplified from template DNA using forward and reverseprimers designed such that the initial PCR products would containoverlapping sequences, allowing, overlapping PCR to generate the codingsequences of the desired polypeptide constructs.

Initial PCR amplification of template DNA: Approximately 35 ng oftemplate DNA, e.g. light chain and heavy chain of antibody of interest;1 ul of 10 uM forward and reverse primers; 2.5 ul of 10×pfuUltra buffer(Stratagene, Inc.); 1 ul of 10 mM dNTP; 1 ul of 2.5 units/ul of pfuUltraDNA polymerase (Stratagene, Inc.); and distilled water to 25 ul totalvolume were gently mixed in a microfuge tube and briefly spun in amicrocentrifuge to collect the reaction mixture at the bottom of thetube. PCR reactions were performed using GeneAmp PCR System 9700 (PEApplied Biosystem) and the following settings: 94° C., 2 minutes; 25cycles of 94° C., each 15 seconds; 58° C., 30 seconds; and 72° C., 1minute.

The VL of Hu2B6 was amplified from the light chain of Hu2B6 usingforward and reverse primers SEQ ID NO: 55 and SEQ ID NO: 56,respectively. The VH of Hu2B6 was amplified from the heavy chain ofHu2B6 using forward and reverse primers SEQ ID NO: 57 and SEQ ID NO: 58,respectively. The VL of Hu3G8 was amplified from the light chain ofHu3G8 using forward and reverse primers SEQ ID NO: 55 and SEQ ID NO: 59,respectively. The VH of Hu3G8 was amplified from the heavy chain ofHu3G8 using forward and reverse primers SEQ ID NO: 60 and SEQ ID NO: 61,respectively.

PCR products were electrophoresed on a 1% agarose gel for 30 minutes at120 volts. PCR products were cut from the gel and purified usingMinElute GEI Extraction Kit (Qiagen, Inc.).

Overlapping PCR: Initial PCR products were combined as described belowand amplified using the same PCR conditions described for initialamplification of template DNA. Products of overlapping PCR were alsopurified as described supra.

The nucleic acid sequence encoding construct 1, SEQ ID NO: 9 (shownschematically in FIG. 2), was amplified by combining the PCR products ofthe amplifications of VL Hu2B6 and VH Hu3G8, and forward and reverseprimers SEQ ID NO: 55 and SEQ ID NO: 61, respectively. The nucleic acidsequence encoding construct 2, SEQ ID NO: 11 (shown schematically inFIG. 2), was amplified by combining the PCR products of theamplifications of VL Hu3G8 and VH Hu2B6, and forward and reverse primersSEQ ID NO: 55 and SEQ ID NO: 58, respectively. The nucleic acid sequenceencoding construct 3, SEQ ID NO: 12 (shown schematically in FIG. 2), wasamplified by combining the PCR products of the amplifications of VLHu3G8 and VH Hu3G8, and forward and reverse primers SEQ ID NO: 55 andSEQ ID NO: 61, respectively. The nucleic acid sequence encodingconstruct 4, SEQ ID NO: 13 (shown schematically in FIG. 2), wasamplified by combining the PCR products of the amplifications of VLHu2B6 and VH Hu2B6, and forward and reverse primers SEQ ID NO: 55 andSEQ ID NO: 58, respectively.

The forward primers of the VL domains (i.e., SEQ ID NO: 55) and reverseprimers of the VH domains (i.e., SEQ ID NO: 58 and SEQ ID NO: 61)contained unique restriction sites to allow cloning of the final productinto an expression vector. Purified overlapping PCR products weredigested with restriction endonucleases Nhe I and EcoR I, and clonedinto the pCIneo mammalian expression vector (Promega, Inc.). Theplasmids encoding constructs were designated as identified in Table 12:

TABLE 12 PLASMID CONSTRUCTS Encoding Construct Plasmid DesignationInsert 1 pMGX0669 hu2B6VL-hu3G8VH 2 pMGX0667 hu3G8VL-hu2B6VH 3 pMGX0666hu3G8VL-hu3G8VH 4 pMGX0668 hu2B6VL-hu2B6VH

Polypeptide/diabody Expression: pMGX0669, encoding construct 1, wascotransfected with pMGX0667, encoding construct 2, in HEK-293 cellsusing Lipofectamine 2000 according to the manufacturer's directions(Invitrogen). Co-transfection of these two plasmids was designed to leadto the expression of a covalent bispecific diabody (CBD) immunospecificfor both FcγRIIB and FcγRIIIA (the h2B6-h3G8 diabody). pMGX0666 andpMGX0668, encoding constructs 3 and 4, respectively, were separatelytransfected into HEK-293 cells for expression of a covalent monospecificdiabody (CMD), immunospecific for FcγRIIIA (h3G8 diabody) and FcγRIIB(h2B6 diabody), respectively. Following three days in culture, secretedproducts were purified from the conditioned media.

Purification: Diabodies were captured from the conditioned medium usingthe relevant antigens coupled to CNBr activated Sepharose 4B. Theaffinity Sepharose resin was equilibrated in 20 mM Tris/HCl, pH 8.0prior to loading. After loading, the resin was washed with equilibrationbuffer prior to elution. Diabodies were eluted from the washed resinusing 50 mM Glycine pH 3.0. Eluted diabodies were immediatelyneutralized with 1M Tris/HCl pH 8.0 and concentrated using acentrifugation type concentrator. The concentrated diabodies werefurther purified by size exclusion chromatography using a Superdex 200column equilibrated in PBS.

SEC: Size exclusion chromatography was used to analyze the approximatesize and heterogeneity of the diabodies eluted from the column. SECanalysis was performed on a GE healthcare Superdex 200HR 10/30 columnequilibrated with PBS. Comparison with the elution profiles of a fulllength IgG (˜150 kDa), an Fab fragment (˜50 kDa) and a single chain Fv(˜30 kDa) were used as controls).

ELISA: The binding of eluted and purified diabodies was characterized byELISA assay, as described in 5.4.2. 50 ul/well of a 2 ug/ml solution ofsCD32B-Ig was coated on 96-well Maxisorp plate in Carbonate buffer at 4°C. over night. The plate was washed three times with PBS-T (PBS, 0.1%Tween 20) and blocked by 0.5% BSA in PBS-T for 30 minutes at roomtemperature. Subsequently, h2B6-h3G8 CBD, h2B6 CMD, or h3G8 CMD werediluted into the blocking buffer in a serial of two-fold dilutions togenerate a range of diabody concentrations, from 0.5 μg/ml to 0.001μg/ml. The plate was then incubated at room temperature for 1 hour.After washing with PBS-T three times, 50 μl/well of 0.2 ug/mlsCD16A-Biotin was added to each well. The plate was again incubated atroom temperature for 1 hour. After washing with PBS-T three times, 50μl/well of a 1:5000 dilution of HRP conjugated streptavidin (AmershamPharmacia Biotech) was used for detection. The HRP-streptavidin wasallowed to incubate for 45 minutes at room temperature. The plate waswashed with PBS-T three times and developed using 80 μl/well of TMBsubstrate. After a 10 minute incubation, the HRP-TMB reaction wasstopped by adding 40 ul/well of 1% H₂SO₄. The OD450 nm was read by usinga 96-well plate reader and SOFTmax software, and results plotted usingGraphPadPrism 3.03 software.

BIAcore Assay: The kinetic parameters of the binding of eluted andpurified diabodies were analyzed using a BIAcore assay (BIAcoreinstrument 1000, BIAcore Inc., Piscataway, N.J.) and associated softwareas described in section 5.4.3.

sCD16A, sCD32B or sCD32A (negative control) were immobilized on one ofthe four flow cells (flow cell 2) of a sensor chip surface through aminecoupling chemistry (by modification of carboxymethyl groups with mixtureof NHS/EDC) such that about 1000 response units (RU) of either receptorwas immobilized on the surface. Following this, the unreacted activeesters were “capped off” with an injection of 1M Et-NH2. Once a suitablesurface was prepared, covalent bispecific diabodies (h2B6-h3G8 CBD) orcovalent monospecific diabodies (h2B6 CMD or h3G8 CMB) were passed overthe surface by 180 second injections of a 6.25-200 nM solution at a 70mL/min flow rate. h3G8 scFV was also tested for comparison.

Once an entire data set was collected, the resulting binding curves wereglobally fitted using computer algorithms supplied by the manufacturer,BIAcore, Inc. (Piscataway, N.J.). These algorithms calculate both theK_(on) and K_(off), from which the apparent equilibrium bindingconstant, K_(D) is deduced as the ratio of the two rate constants (i.e.,K_(off)/K_(on)). More detailed treatments of how the individual rateconstants are derived can be found in the BIAevaluaion Software Handbook(BIAcore, Inc., Piscataway, N.J.).

Association and dissociation phases were fitted separately. Dissociationrate constant was obtained for interval 32-34 sec of the 180 secdissociation phase; association phase fit was obtained by a 1:1 Langmuirmodel and base fit was selected on the basis R_(max) and chi² criteriafor the bispecific diabodies and scFv; Bivalent analyte fit was used forCMD binding.

Results

SDS-PAGE analysis under non-reducing conditions revealed that thepurified product of the h3G8 CMD, h2B6 CMD and h2B6-h3G8 CBD expressionsystems were each a single species with an estimated molecular weight ofapproximately 50 kDa (FIG. 3, lanes 4, 5 and 6, respectively). Underreducing conditions, the product purified from either of the CMDexpression systems ran as a single band (lanes 1 and 2), while theproduct purified from the h2B6-h3G8 CBD system was revealed to be 2separate proteins (FIG. 3, lane 3). All polypeptides purified from theexpression system and visualized by SDS-PAGE under reducing conditionsmigrated at approximately 28 kDa.

SEC analysis of each of the expression system products also revealed asingle molecular species (FIG. 4B), each of which eluted at the sameapproximate time as an Fab fragment of IgG (˜50 kDa) (FIG. 4A). Theresults indicate that affinity purified product was a homogenouscovalent homodimer for the case of CMD expression system and ahomogenous covalent heterodimer for the case of the h2B6-h3G8 CBD.

An ELISA sandwich assay was used to test binding of the h2B6-h3G8 CBDfor specificity to either or both of CD32B and/or CD16A (FIG. 5). CD32Bserved as the target antigen and CD16A was used as the secondary probe.The positive signal in the ELIZA revealed that the heterodimerich2B6-h3G8 CBD had specificity for both antigens. Similar testing of theh3G8 CMD (which should not bind CD32B) showed no signal.

SPR analysis indicated that h3G8 CMD immunospecifically recognized sCD16but not sCD32B, that h2B6 CMD immunospecifically recognized sCD32B butnot sCD16, and that h2B6-h3G8 CBD immunospecifically recognized bothsCD16 and sCD32B (FIGS. 6A-B). None of the diabodies tested bound thecontrol receptor, sCD32A (FIG. 6C).

SPR analysis was also used to estimate the kinetic and equilibriumconstants of the CMDs and h2B6-h3G8 CBD to sCD16 and/or sCD32B. Resultswere compared to the same constants calculated for an h3G8 scFV. FIGS.7A-E show the graphical results of the SPR analysis. The kinetic on andoff rates, as well as the equilibrium constant, calculated from theresults depicted in FIG. 7 are provided in Table 13.

TABLE 13 Kinetic and Equilibrium Constants Calculated from BIAcore Data.Receptor/Analyte k-on k-off Kd sCD16/h3G8 diabody 2.3 × 10⁵ 0.004 18.0sCD16/h2B6-h3G8 CBD 4.6 × 10⁵ 0.010 22.7 sCD16/h3G8 scFv 3.2 × 10⁵ 0.01338.7 sCD32B/h2B6-h3G8 CBD 3.6 × 10⁵ 0.005 15.0 sCD32B/h2B6 diabody 6.2 ×10⁵ 0.013 21.0

Coupled with the results of the ELISA analysis, the studies confirm thatthe h2B6-h3G8 covalent heterodimer retained specificity for both CD32Band CD16, and was capable of binding both antigens simultaneously. Themolecule is schematically represented in FIG. 8.

6.2 Design and Characterization of Covalent Bispecific DiabodiesComprising Fc Domains

In an effort to create an IgG like molecule, i.e., comprising an Fcdomain, one of the polypeptides comprising the heterodimeric CBDmolecule presented in Example 6.1 was modified to further comprise an Fcdomain (creating a ‘heavier’ and ‘lighter’ chain, analogous to anantibody heavy and light chain). The heterodimeric bispecific moleculewould then contain an Fc domain that will dimerize with a homologousmolecule, forming a tetrameric IgG-like molecule with tetravalency (i.e,formed by dimerization via the Fc domains of the heterodimericbispecific molecules). Interestingly, such tetrameric molecules were notdetected in the conditioned media of recombinant expression systemsusing functional assays, e.g., testing the conditioned media forimmunospecific binding to target antigens. Instead, only a dimericmolecule, comprising monomers consisting of a VL, VH and Fc domain, weredetected in such functional assays. To test whether stability of thetheoretical tetrameric structure was at issue, polypeptides comprisingthe Fc domain were engineered to further comprise a hinge region whilethe polypeptides comprising the ‘lighter’ chain were engineered tofurther comprise the 6 C-terminal amino acids of the constant domain ofthe human kappa light chain. When such reengineered ‘heavier’ and‘lighter’ chains were co-expressed in the recombinant expressionsystems, functional assays detected diabody molecules that were able toimmunospecifically bind both of the target antigens and anti-Fcantibodies.

Materials and Methods

Construction and Design of Polypeptide Molecules: Nucleic acidexpression vectors were designed to produce modified versions ofconstructs 1 and 2 presented in Example 6.1. Construct 5 (SEQ ID NO: 14)and 6 (SEQ ID NO: 15), were created by engineering construct 1 and 2,respectively to further comprise an Fc domain. Construct 7 (SEQ ID NO:16) was created by engineering construct 1 was to further comprise thesequence FNRGEC (SEQ ID NO: 23) at its C-terminus. Construct 8 (SEQ IDNO: 18) was created by engineering construct 2 to further comprise ahinge region and Fc domain (comprising V215A mutation). Schematicrepresentation of constructs 5-8 is shown in FIG. 9.

PCR and Expression Vector Construction: All PCR and PCR productpurification protocols were as described in Example 6.1 PlasmidspMGX0669 and pMGX0667 served as templates for the coding sequences ofconstructs 1 and 2, respectively. The coding sequences for the of HuIgGFc domain and/or hinge domain were SEQ ID NO: 5 or SEQ ID NO: 1 and SEQID NO: 5, respectively. The coding sequences of the template DNAs wereamplified using forward and reverse primers such that the PCR productswould contain overlapping sequences, allowing overlapping PCR togenerate the coding sequences of the desired products.

The coding sequence of construct 1 was amplified from pMGX0669 usingforward and reverse primers SEQ ID NO: 55 and SEQ ID NO: 62,respectively. The coding sequence of construct 2 was amplified frompMGX0667 using forward and reverse primers SEQ ID NO: 55 and SEQ ID NO:63, respectively. HuIgG hinge-Fc was amplified using forward and reverseprimers SEQ ID NO: 65 and SEQ ID NO: 66, respectively. Construct 7 (SEQID NO: 16) was amplified from pMGX0669 using forward and reverse primersSEQ ID NO: 55 and SEQ ID NO: 67.

Overlapping PCR: Initial PCR products were combined as described below,amplified and purified as described in example 6.1.

The nucleic acid sequence encoding construct 5, SEQ ID NO: 14 (shownschematically in FIG. 9), was amplified by combining the PCR products ofthe amplifications of construct 1 and HuIgG Fc, and forward and reverseprimers SEQ ID NO: 55 and SEQ ID NO: 64, respectively. The nucleic acidsequence encoding construct 6, SEQ ID NO: 15 (shown schematically inFIG. 9), was amplified by combining the PCR products of theamplifications of construct 2 and HuIgG Fc, and forward and reverseprimers SEQ ID NO: 55 and SEQ ID NO: 66, respectively. The nucleic acidsequence encoding construct 8, SEQ ID NO: 18 (shown schematically inFIG. 9), was amplified by combining the PCR products of theamplifications of construct 2 and HuIgG hinge-Fc, and forward andreverse primers SEQ ID NO: 55 and SEQ ID NO: 66, respectively.

Final products were cloned into pCIneo mammalian expression vector(Promega, Inc.) as previously described. The plasmid encoding constructswere designated as identified in Table 14:

TABLE 14 PLASMID CONSTRUCTS Encoding Plasmid Construct DesignationInsert 5 pMGX0676 hu2B6VL-hu3G8VH-huFc 6 pMGX0674 hu3G8VL-hu2B6VH-huFc 7pMGX0677 Hu2B6VL-hu3G8VH- FNRGEC 8 pMGX0678 Hu3G8VL-hu2B6VH-huhinge- Fc(A215V)

Polypeptide/diabody Expression: Four separate cotransfections into inHEK-293 cells using Lipofectamine 2000, as described in section 6.1,were performed: pMGX0669 and pMGX0674, encoding constructs 1 and 6,respectively; pMGX0667 and pMGX0676, encoding constructs 2 and 5,respectively; and pMGX0677 and pMGX0678, encoding constructs 7 and 8,respectively.

Co-transfection of these plasmids was designed to lead to the expressionof a bispecific diabody (CBD) of tetravalency with IgG-like structure,immunospecific for both FcγRIIB and FcγRIIIA. An additionalcotransfection was also performed: pMGX0674 and pMGX0676, encodingconstructs 6 and 5, respectively. Following three days in culture,conditioned media was harvested. The amount of secreted product in theconditioned media was quantitated by anti IgG Fc ELISA using purified Fcas a standard. The concentrations of product in the samples was thennormalized based on the quantitation, and the normalized samples usedfor the remaining assays.

ELISA: The binding of diabody molecules secreted into the medium wasassayed by sandwich ELISA as described, supra. Unless indicated, CD32Bwas used to coat the plate, i.e., as the target protein, andHRP-conjugated CD16 was used as the probe.

Results

An ELISA assay was used to test the normalized samples from therecombinant expression systems comprising constructs 1 and 6(pMGX669-pMGX674), constructs 2 and 5 (pMGX667-pNGX676) and constructs 5and 6 (pMGX674-pMGX676) for expression of diabody molecules capable ofsimultaneous binding to CD32B and CD16A (FIG. 10). The ELISA dataindicated that co-transfection with constructs 1 and 6 orco-transfection with constructs 2 and 5 failed to produce a product thatcould bind either or both antigens (FIG. 10, ▭ and ▴, respectively).However, co-transfection of constructs 5 and 6 lead to secretion of aproduct capable of binding to both CD32B and CD16 antigens. The latterproduct was a dimer of constructs 5 and 6, containing one binding sitefor each antigen with a structure schematically depicted in FIG. 11.

In order to drive formation of an IgG like heterotetrameric structure,the coding sequence for six additional amino acids was appended to theC-terminal of construct 1, generating construct 7 (SEQ ID NO: 16 andshown schematically in FIG. 9). The six additional amino acids, FNRGEC(SEQ ID NO: 23), were derived from the C-terminal end of the Kappa lightchain and normally interact with the upper hinge domain of the heavychain in an IgG molecule. A hinge domain was then engineered intoconstruct 6, generating construct 8 (SEQ ID NO: 18 and FIG. 9).Construct 8 additionally comprised an amino-acid mutation in the upperhinge region, A215V. Expression plasmids encoding construct 7 andconstruct 8, pMGX677 and pMGX678, respectively, were then cotransfectedinto HEK-293 cells and expressed as described.

Diabody molecules produced from the recombinant expression systemcomprising constructs 7 and 8 (pMGX0677+pMGX0678), were compared in anELISA assay for binding to CD32B and CD16A to diabody molecules producedfrom expression systems comprising constructs 1 and 6 (pMGX669+pMGX674),constructs 2 and 8 (pMGX669+pMGX678), and constructs 6 and 7(pMGX677+pMGX674) (FIG. 12).

As before, the molecule produced by the expression system comprisingconstructs 1 and 6 (pMGX669+pMGX674) proved unable to bind both CD32Aand CD16A (FIG. 10 and FIG. 12). In contrast, the product from theco-expression of either constructs 7 and 6 (pMGX0677+pMGX0674) or fromthe co-expression of constructs 7 and 8 (pMGX0677-pMGX0678) were able tobind both CD32B and CD16 (FIG. 12). It is noted that construct 7 isanalogous to construct 1, with the exception that construct 7 comprisesthe C-terminal sequence FNRGEC (SEC ID NO:23); and that construct 8 isanalogous to construct 6, except that construct 8 comprises a hingedomain and the mutation A215V. The data indicate that the addition ofthe 6 extra amino-acids from the C-terminus of the C-kappa light chain(FNRGEC; SEQ ID NO: 23) to the non-Fc bearing, ‘lighter,’ chain helpedstabilize the formation of the tetrameric IgG-like diabody molecules,regardless of whether the corresponding heavier chain comprised a hingedomain (i.e., pMGX0677+pMGX0674 and pMGX0677-pMGX0678, FIG. 12). Theaddition of the hinge domain to the Fc bearing ‘heavier’ polypeptide,without the addition of the FNRGEC (SEQ ID NO: 23) C-terminal sequenceto the corresponding ‘lighter’ chain, was apparently unable to effectsimilar stabilization (i.e., lack of binding by product ofco-transfection of constructs 2 and 8 (pMGX669+pMGX678)). The structureof the tetrameric diabody molecule is schematically represented in FIG.13.

6.3 Effect of Domain Order and Additional Disulfide Bonds on Formationof Tetrameric IgG-Like Diabody

The effect of additional stabilization between the ‘lighter’ and‘heavier’ polypeptide chains of the tetrameric IgG-like diabody moleculewas investigated by substitution of selected residues on the polypeptidechains with cysteines. The additional cysteine residues provide foradditional disulfide bonds between the ‘heavier’ and ‘lighter’ chains.Additionally, domain order on binding activity was investigated bymoving the Fc domain or the hinge-Fc domain from the C-terminal end ofthe polypeptide chain to the N-terminus. Although the binding activityof the molecule comprising the additional disulfide bonds was notaltered relative to earlier constructed diabody molecules with suchbonds, transferring the Fc or hinge-Fc domain to the N-terminus of the‘heavier’ polypeptide chain comprising the diabody surprisingly improvedbinding affinity and/or avidity of the bispecific molecule to one orboth of its target antigens.

Materials and Methods

Construction and Design of Polypeptide Molecules: Nucleic acidexpression vectors were designed to produce modified versions ofconstructs 5, 6 and 8 presented in Example 6.2. Construct 9 (SEQ ID NO:19) and construct 10 (SEQ ID NO: 20) (both shown schematically in FIG.13) were analogous to constructs 8 and 6, with the exception that Fcdomain or hinge-Fc domain, respectively, was shifted from the C-terminusof the polypeptide to the N-terminus. Additionally all Fc domains usedwere wild-type IgG1 Fc domains. Construct 11, SEQ ID NO: 21, (shownschematically in FIG. 14) was analogous to construct 2 from Example 6.1except that the C-terminus was designed to further comprise the sequenceFNRGEC (SEQ ID NO: 23). Construct 12, SEQ ID NO: 22 (shown schematicallyin FIG. 14) was analogous to construct 5 from Example 6.2 except thatthe Fc domain further comprised a hinge region: Also, for constructs 11and 12, the 2B6 VL domain and 2B6 VH domain comprised a single aminoacid modification (G105C and G44C, respectively) such that a glycine ineach domain was replaced by cysteine.

PCR and Expression Vector Construction: All PCR and PCR productpurification protocols were as described in Example 6.1 and 6.2

Overlapping PCR: Final products were constructed, amplified and purifiedusing methods described in example 6.1 and example 6.2.

Final products were cloned into pCIneo mammalian expression vector(Promega, Inc.) as previously described. The plasmid encoding constructswere designated as identified in Table 15:

TABLE 15 PLASMID CONSTRUCTS Encoding Construct Plasmid DesignationInsert 9 pMGX0719 Huhinge/Fc-hu3G8VL- hu2B6VH 10 pMGX0718 HuFc-hu2B6VL-hu3G8VH 11 pMGX0716 Hu2B6VL(G/C)- hu3G8VH-huhingeFC 12 pMGX0717Hu3G8VL-hu2B6VH (G/C)-FNRGEC

Polypeptide/diabody Expression: Three separate cotransfections in to inHEK-293 cells using Lipofectamine 2000, as described in section 6.1,were performed: pMGX0669 and pMGX0719, encoding constructs 1 and 9,respectively; pMGX0669 and pMGX0718, encoding constructs 1 and 10,respectively; and pMGX0617 and pMGX0717, encoding constructs 11 and 12,respectively. Co-transfection of these plasmids was designed to lead tothe expression of a bispecific diabody (CBD) of tetravalency withIgG-like structure, immunospecific for both FcγRIIB and FcγRIIIA.Following three days in culture, conditioned media was harvested. Theamount of secreted product in the conditioned media was quantitated byanti IgG Fc ELISA using purified Fc as a standard. The concentrations ofproduct in the samples were then normalized based on the quantitation,and the normalized samples used for the remaining assays.

ELISA: The binding of diabody molecules secreted into the medium wasassayed by sandwich ELISA as described, supra. Unless indicated, CD32Bwas used to coat the plate, i.e., as the target protein, andHRP-conjugated CD16 was used as the probe.

Western Blot: Approximately 15 ml of conditioned medium form the threeabove-described cotransfections were analyzed by SDS-PAGE undernon-reducing conditions. One gel was stained with Simply Blue Safestain(Invitrogen) and an identical gel was transferred to PVDF membrane(Invitrogen) using standard transfer methods. After transfer, themembrane was blocked with 5% dry skim milk in 1×PBS. The membrane wasthen incubated in 10 ml of 1:8,000 diluted HRP conjugated Goat antihuman IgG1 H+L in 2% dry skim milk 1×PBS/0.1% Tween 20 at roomtemperature for 1 hr with gentle agitation. Following a wash with1×PBS/0.3% Tween 20, 2×5 min each, then 20 min at room temperature, themembrane was developed with ECL Western blotting detection system(Amersham Biosciences) according to the manufacturer's instructions. Thefilm was developed in X-ray processor.

Results

Conditioned media from the recombinant expression systems comprisingconstructs 1 and 9; constructs 1 and 10; and constructs 11 and 12 wereanalyzed by SDS-PAGE (under non reducing conditions) analysis andWestern-blotting (using an anti-IgG as the probe). Western blot revealedthat the product from the systems comprising constructs 11 and 12 orcomprising constructs 9 and 1 predominately formed a single species ofmolecule of approximately 150 kDa (FIG. 14, lanes 3 and 2,respectively). Both of these products have engineered internal disulfidebonds between the ‘lighter’ and ‘heavier’ chains comprising the diabody.In contrast, the molecule without engineered internal disulfide bondsbetween the ‘lighter’ and ‘heavier’ chains, formed of constructs 10 and1, formed at least two molecular species of molecular weights ˜75 and˜100 kDa (FIG. 14, lane 1).

Despite the results of the Western Blot, each of the three products wasfound capable of binding both CD32A and CD16 (FIG. 15). Surprisingly,relative to the product comprising a C-terminal hinge-Fc domain (formedof constructs 11 and 12), the product from both systems wherein the Fc(or Fc-hinge) domain was at the amino terminus of the Fc containingpolypeptide chain (i.e., the ‘heavier’ chain) (constructs 9+1 andconstructs 10+1) demonstrated enhanced affinity and/or avidity to one orboth of its target peptides (i.e. CD32B and/or CD16).

6.4 Effect of Internal/External Cleavage Site on Processing ofPolyprotein Precursor and Expression of Covalent Bispecific Diabody;Design and Characterization of Bispecific Diabody Comprising Portions ofHuman IgG Lambda Chain and Hinge Domain

As described herein, the individual polypeptide chains of the diabody ordiabody molecule of the invention may be expressed as a singlepolyprotein precursor molecule. The ability of the recombinant systemsdescribed in Examples 6.1-6.3 to properly process and express afunctional CBD from such a polyprotein precursor was tested byengineering a nucleic acid to encode, both the first and secondpolypeptide chains of a CBD separated by an internal cleavage site, inparticular, a furin cleavage site. Functional, CBD was isolated from therecombinant system comprising the polyprotein precursor molecule.

As discussed in Example 6.3, addition of the 6 C-terminal amino acidsfrom the human kappa light chain, FNRGEC (SEQ ID NO: 23), was found tostabilize diabody formation—presumably through enhanced inter-chaininteraction between the domains comprising SEQ ID NO: 23 and thosedomains comprising an Fc domain or a hinge-Fc domain. The stabilizingeffect of this lambda chain/Fc like interaction was tested in CBDwherein neither polypeptide chain comprised an Fc domain. Onepolypeptide chain of the diabody was engineered to comprise SEQ ID NO:23 at its C-terminus; the partner polypeptide chain was engineered tocomprise the amino acid sequence VEPKSC (SEQ ID NO: 77), which wasderived from the hinge domain of an IgG. Comparison of this CBD to thatcomprised of constructs 1 and 2 (from example 6.1) revealed that the CBDcomprising the domains derived from hinge domain and lambda chainexhibited slightly greater affinity to one or both of its targetepitopes.

Materials and Methods

-   -   Construction and Design of Polypeptide Molecules: Polyprotein        precursor: Nucleic acid expression vectors were designed to        produce 2 polyprotein precursor molecules, both represented        chematically in FIG. 17. Construct 13 (SEQ ID NO: 95) comprised        from the N-terminus of the polypeptide chain, the VL domain of        3G8, the VH domain of 2.4G2 (which binds mCD32B), a furin        cleavage site, the VL domain of 2.4G2 and the VH domain of 3G8.        The nucleotide sequence encoding construct 13 is provided in SEQ        ID NO: 96. Construct 14 (SEQ ID NO: 97) (FIG. 17), comprised        from the N-terminus of the polypeptide chain, the VL domain of        3G8, the VH domain of 2.4G2 (which binds mCD32B), a furin        cleavage site, a FMD (Foot and Mouth Disease Virus Protease C3)        site, the VL domain of 2.4G2 and the VH domain of 3G8. The        nucleotide sequence encoding construct 14 is provided in SEQ ID        NO: 98.

Nucleic acid expression vectors were designed to produce modifiedversions of constructs 1 and 2 presented in Example 6.1. Construct 15(SEQ ID NO: 99) (FIG. 17) was analagous to construct 1 (SEQ ID NO: 9),presented in example 6.1, with the exception that the C-terminus ofconstruct 15 comprised the amino acid sequence FNRGEC (SEQ ID NO: 23).The nucleic acid sequence encoding construct 15 is provided in SEQ IDNO: 100. Construct 16 (SEQ ID NO: 101) (FIG. 17) was analogous toconstruct 2, presented in Example 6.1, with the exception that theC-terminus of construct 16 comprised the amino acid sequence VEPKSC (SEQID NO: 77). The nucleic acid sequence encoding construct 16 is providedin SEQ ID NO: 102.

PCR and Expression Vector Construction: All PCR and PCR productpurification protocols were as described in Example 6.1 and 6.2

Overlapping PCR: Final products were constructed, amplified and purifiedusing methods described in example 6.1 and example 6.2 with appropriateprimers

Final products were cloned into pCIneo mammalian expression vector(Promega, Inc.) as previously described. The plasmid encoding constructswere designated as identified in Table 16:

TABLE 16 PLASMID CONSTRUCTS Encoding Construct Plasmid DesignationInsert 13 pMGX0750 3G8VL-2.4G2VH-Furin- 2.4G2VL-3G8VH 15 pMGX0752Hu2B6VL-Hu3G8VH- FNRGEC 16 pMGX0753 Hu3G8VL-Hu2B6VH- VEPKSC

Polypeptide/diabody Expression: One transfection and one cotransfectioninto in HEK-293 cells using Lipofectamine 2000, as described in section6.1, were performed: single: pMGX0750, encoding construct 13; andcotransfection: pMGX0752 and pMGX0753, encoding constructs 15 and 16,respectively. Following three days in culture, conditioned media washarvested, and secreted product affinity purified as described.

ELISA: The binding of diabody molecules secreted into the medium wasassayed by sandwich ELISA as described, supra. Murine CD32B was used tocoat the plate, i.e., as the target protein, and HRP-conjugated CD16Awas used as the probe for the product of the co-transfection ofconstructs 15 and 16. mCD32B was used as the target protein andbiotin-conjugated CD16A was used as the probe for the recombinant systemcomprising construct 13.

Results

Conditioned media from the recombinant expression systems comprisingconstructs 13 was analysed by sandwich ELISA. The ELISA assay tested thebinding of the CBD for specificity to either or both of mCD32B and/orCD16 (FIG. 18). CD32B served as the target antigen and CD16A was used asthe secondary probe. The positive signal in the ELISA revealed that theheterodimeric h2.4G2-h3G8 CBD produced from the polyprotein precursorhad specificity for both antigens.

Similarly, the purified product generated by cotransfection of thevectors encoding constructs 15 and 16 was tested in an ELISA assay andcompared to the product comprised of constructs 1 and 2 (Example 6.1).CD32B served as the target antigen and CD16A was used as the secondaryprobe. As with the product comprised of constructs 1 and 2, the productof constructs 15 and 16 was found to be capable of simultaneouslybinding CD32B and CD16A. In fact, the product of constructs 15 and 16showed slightly enhanced affinity for one or both of the targetantigens, i.e. CD32B or CD16A. This is perhaps due to increasedstability and or fidelity (relative to a wild type VH-VL domaininteraction) of the interchain association afforded by the interactionof the lambda chain region, FNRGEC (SEQ ID NO: 23) and hinge regionVEPKSC (SEQ ID NO: 77), which is absent in the product comprised ofconstructs 1 and 2.

6.5 Use of Dual Affinity Retargeting Reagents (“DARTs”) to Link MultipleAffinities Together

One aspect of the present invention relates to new dual affinityretargeting reagents (“DARTs”) as well as new ways of linking multipleaffinities together. “DARTS” may be monospecific, bispecific,trispecific, etc., thus being able to simultaneously bind one, two,three or more different epitopes (which may be of the same or ofdifferent antigens). “DARTS” may additionally be monovalent, bivalent,trivalent, tetravalent, pentavalent, hexavelent, etc., thus being ableto simultaneously bind one, two, three, four, five, six or moremolecules. As shown in FIG. 35, these two attributes of DARTS may becombined, for example to produce bispecific antibodies that aretetravalent, etc.

One advance is the development of a DART that has affinity for aprototypic immune receptor, huCD32B, as well as affinity for a hapten,fluorescein. This DART, termed “2B6/4420,” serves as a universaladaptor, able to co-ligate huCD32B with molecules that interacts withfluorescein-conjugated binding partners. CD32B is an Fc receptor thathas the ability to quench activating signals by virtue of clusteringwith activation signaling immune complexes. In its initialimplementation, this technology allows rapid screening of severalbiological targets for clustering with huCD32B without the need togenerate new DART constructs. The 2B6/4420 can simply be mixed with afluoresceinated antibody against a cell surface receptor and therebymimic the action of a DART with affinity for that receptor (FIG. 20).Further, this reagent allows efficient linkage of affinity reagents thatare not easily expressed or produced, allowing one to overcome technicallimitations. 2B6/4420-containing DARTs are clearly useful as researchtools and also as clinical candidates. 2B6/4420 produced from HEK293cells can simultaneously bind CD32B and fluorescein in an ELISA assay.Additionally, it can inhibit cell proliferation by recruiting CD32B tothe BCR complex via colligation with CD79. The 2B6 arm of the DART maybe replaced with a different antibody sequence or a binding sequencehaving other relevant specificity.

Materials and Methods:

Plasmid Constructs: 2B6/4420 is derived from sequences of humanized 2B6MAb (hu2B6, MGA321) and a chimeric mouse Fv/human Fc version of theanti-fluorescein MAb, 4420. The fully assembled DART consists of twopolypeptides, resulting in covalent linkage of two Fv regions. The firstpolypeptide consists of a secretion signal sequence followed by thehu2B6VL produced as a fusion protein with 4420VH separated by a linkerconsisting of the amino acid residues GGGSGGGG. The sequence FNRGEC,derived from the C-terminus of the kappa light chain, is appended to theC-terminus of this polypeptide. The other polypeptide consists of signalsequence-4420VL-GGGSGGGG-hu2B6VH, with the sequence VEPKSC, derived fromthe C-terminus of the human IgG1 Fd fragment, appended to theC-terminus. The cysteines in the two chains form a disulfide bond,covalently linking the two polypeptides together (FIG. 20). The DNAsequences encoding the described polypeptides were PCR amplified fromexisting plasmids, combined by overlap PCR and cloned into pCIneo(Promega) between the Nhe I and EcoR I sites. Finally, a DART withaffinity for huCD32B and huCD16 (2B6/3G8) that has been previouslyconstructed using methods similar to those described above was used as acontrol.

Antibodies: The murine monoclonal antibodies anti-human CD79b, CB3.1 andCB3.2 (hybridomas) were obtained from Dr. Cooper MD, University ofAlabama at Birmingham, Birmingham Ala. CB3.1 and CB3.2 were labeled withfluorescein isothiocyanate (FITC) following the manufacturerinstructions (Pierce, Rockford Ill.). The F(ab′)₂ fragment of anFe-fragment-specific, goat anti-mouse (GAM) IgG was obtained fromJackson Laboratories (West Grove, Pa.). Anti-huCD32B mouse MAb, 3H7, wasproduced and purified in house. Goat anti-2B6Fv was produced byimmunizing goats with hu2B6 whole antibody and affinity purifyingagainst the Fv region of hu2B6. HulgG, FITC-hulgG, and HRP-anti-mouseIgG were obtained from Jackson Immunoresearch. HRP-anti-goat wasobtained from Southern Biotech.

DART expression: Plasmids encoding each chain were cotransfected into293H cells (Invitrogen) using Lipofectamine 2000 (Invitrogen) accordingto the manufacturer's instructions. Secreted protein was harvested 3-4times at three day intervals and purified by liquid chromatographyagainst an immobilized soluble form of CD32B.

ELISA: 2B6/4420 or 2B6/3G8 DARTs were captured on MaxiSorp plates (NalgeNunc) coated with FITC-labeled Protein S (Novagen), human IgG, orFITC-huIgG. Detection proceeded by binding soluble CD32B ectodomain,followed by 3H7 (a mouse monoclonal antibody specific for CD32B), andfinally anti-mouse-HRP. Alternatively, detection was performed bybinding goat anti-2B6 Fv polyclonal affinity purified antiserum,followed by anti-goat-HRP. HRP activity was detected using acolorimetric TMB substrate (BioFX) and read on a VersaMax ELISA platereader.

B Cell Purification and Proliferation Assay: Peripheral bloodmononuclear cells were separated by a Ficoll/Paque Plus (AmershamPharmacia Biotech, UK) gradient method using blood from healthy donors.B lymphocytes were isolated using Dynal B Cell Negative Isolation Kit(Dynal Biotechnology Inc., NY) following the manufacture's instructions.The purity of the isolated B cells (CD20⁺) was greater than 90% asestimated by FACS analysis. For the proliferation assay, purified Bcells were seeded in complete RPMI 1640 medium in flat-bottomed 96-wellmicrotiter plates at a cell density of 1×10⁵ cells per well in a finalvolume of 200 μl and incubated for 48 hrs in the presence or absence ofantibodies and diabodies at 37° C. in 5% CO₂. 1 μCi/well of[³H]thymidine (Perkin Elmer, Wellesley, Mass.) was then added and theincubation continued for an additional 16-18 h prior to harvesting.[³H]thymidine incorporation was measured by liquid scintillationcounting.

Results

In order to demonstrate that 2B6/4420 DART was active and specific, twoELISA experiments were conducted. First, 2B6/4420 or 2B6/3G8 (as anegative control) was bound to a fluorescein-conjugated protein(S-protein) that had been coated onto ELISA plates. Next, the 2B6 arm ofthe DART was engaged by soluble CD32B. Binding was detected by anotherantibody to CD32B with an epitope that does not overlap that of 2B6followed an HRP-conjugated secondary antibody. While 2B6/4420 DART iscapable of simultaneously binding fluorescein and CD32B, 2B6/3G8 is not(FIG. 21, Panel A). When the DARTs are captured on plates coated withsoluble CD32B and binding is detected by an antibody specific for hu2B6Fv, both DARTS show good binding. To demonstrate that 2B6/4420 DART wascapable of binding fluorescein conjugated to human IgG (given that thisis the context of the initial implementation of this reagent), HuIgG,unlabeled or labeled with fluorescein, was bound to ELISA plates andused to capture 2B6/4420. Again, 2B6/3G8 was used as a negative control.Binding was detected using an antibody specific for Hu2B6 Fv. 2B6/4420DART clearly binds to FITC-HuIgG, but does not bind to unlabeled HuIgG,demonstrating that this DART is capable of binding fluoresceinconjugated to an antibody and that there is no significant binding toantibody alone. As expected, no binding was detected by 2B6/3G8 DART ineither of these contexts.

Experiments were conducted to demonstrate that the 2B6/4420 DART wascapable of functioning as a dual affinity reagent that could have aneffect upon signaling in the context of a cell-based assay.Co-aggregation of CD32B with the BCR has been shown to inhibit B cellactivation. The ability of the 2B6/4420 DART to co-engage CD32B with theBCR coated with αCD79b antibodies labeled with fluorescein and triggerinhibition of cell proliferation was explored. B cells were negativelyselected from human blood and activated through treatment withincreasing concentrations of mouse anti-human-CD79b FITC-labeled, clonesCB3.1 and CB3.2, and by the addition of a F(ab′)₂ fragment of anFc-specific GAM as a secondary reagent to cross-link the BCR, togetherwith a fixed concentration (5 μg/mL) of 2B6/4420 DART or an equivalentamount of 2B6/3G8 DART, a molecule which does not target fluorescein,thus used as control. Cell proliferation, measured as [³H]-thymidineincorporation, increased with increasing concentrations of themonoclonal anti-CD79b-FITC activator in the absence of DARTS or in thepresence of the control 2B6/3G8 DART. The presence of 2B6/4420 DART ledto a profound reduction in B-cell proliferation at all concentrations ofanti-human CD79b-FITC (FIG. 22, Panels A and B and FIG. 23, Panel A).

Inhibition of proliferation was not observed when B cells coated withunlabeled CB3.2 and activated using the same experimental conditionswere treated with 2B6/4420 DART proving its target-specificity (FIG. 23,Panel B). These data demonstrate that 2B6/4420 DART is able tocross-link CD32B and the BCR and deliver an inhibitory signal capable ofblocking antigen-receptor-induced cell activation.

6.6 DART Immunotherapeutic Against CD32B Expressing B CELL Malignancies

Currently, B cell malignancies are treated using Rituxan® anti-CD20antibody. Some B cell malignancies, however do not express CD20 orbecome resistant to Rituxan. The DARTs of the present invention providean alternative immunotherapeutic capable of overcoming the problemsassociated with Rituxan® anti-CD20 antibody.

MGD261 is a dual-affinity re-targeting (DART) molecule binding to hCD32B(via h2B6 antibody) and hCD16A and hCD16B (via h3G8 antibody).

The efficacy (B cell depletion) and safety of MGD261 was tested inmCD32−/− hCD16A+C57BI/6, mCD32−/− hCD32B+C57BI/6 and mCD32−/−hCD16A+hCD32B+C57BI/6. In this repeat dose experiment, mice received 6IV injections (twice a week for 3 weeks). B cell depletion was monitoredby FACS. Safety was monitored by cage side observation.

Data indicate that MGD261 is capable of depleting B cells in doubletransgenic mice without inducing any significant side effects.

Data: mCD32−/− hCD16A+C57BI/6, mCD32−/− hCD32B+C57BI/6 and mCD32−/−hCD16A+hCD32B+C57BI/6 mice from MacroGenics breeding colony wereinjected IV at days 0, 3, 7, 10, 14 and 17 with MGD261 (10, 3, 1 or 0.3mg/kg), or an irrelevant antibody (hE16 10 mg/kg). Blood was collectedat days—19 (pre-bleed), 4, 11, 18, 25 and 32 for FACS analysis. Animalhealth and activity was recorded three times a week.

Design:

Animals Dose Group # Mice Test Article (mg/kg) A 4 mCD32−/− hCD16A+ hE1610 B 5 mCD32−/− hCD16A+ MGD261 10 C 3 mCD32−/− hCD32B+ hE16 10 D 3mCD32−/− hCD32B+ MGD261 10 E 5 mCD32−/− hCD16A+ hCD32B+ hE16 10 F 5mCD32−/− hCD16A+ hCD32B+ MGD261 10 G 5 mCD32−/− hCD16A+ hCD32B+ MGD261 3H 5 mCD32−/− hCD16A+ hCD32B+ MGD261 1 I 5 mCD32−/− hCD16A+ hCD32B+MGD261 0.3

FACS analysis Method: Whole blood samples were collected at 18 daysprior to h2B6-h3G8 administration and 4, 11, 18, 25 and 32 days afterthe treatment. The blood samples were analyzed to determine the effectof h2B6-h3G8 on the B cell counts by a FACS based assay. A non-washprotocol was used for B cell, T cell and PMN count by using FlowCountbeads, obtained from Beckman Coulter. The panel of antibodies used inthe analysis was 1A8-FITC for PMN, CD3-PE for T cell, CD19-APC for Bcell and CD45-PerCP for total leukocytes.

Results

Mice treated with hE16 or MGD261 (at any concentration) did not show anysign of discomfort at anytime during the duration of theexperimentation.

B cell depletion was observed in hCD16A and hCD32B double transgenicmice. Diabody h2B6-3G8 engages hCD16A expressing effector cells andhCD32B expressing B cells; the engagements were required for the B cellkilling. B cell depletion was not observed in singly transgenic mice(FIG. 24). There were no significant changes for T cells and PMN levelduring the study.

As a further demonstration of the alternative immunotherapeutics of thepresent invention, a surrogate of MGD261, termed “2.4G2-3G8 DB,” wasconstructed. 2.4G2-3G8 DB is a dual-affinity re-targeting (DART)molecule binding to mCD32B (via 2.4G2 antibody) and hCD16A and hCD16B(via h3G8 antibody).

The efficacy (B cell depletion) and safety of 2.4G2-3G8 DB was tested inmCD16−/−, mCD16−/− hCD16A+C57BI/6, mCD16−/− hCD16B+ and mCD16−/−hCD16A+hCD16B+mice. In this repeat dose experiment, mice received 9 IPinjections (Three times a week for 3 weeks). B cell depletion wasmonitored by FACS. Safety was monitored by cage side observation.

Data indicate that 2.4G2-3G8 DB is capable of depleting B cells in hCD16transgenic mice without inducing any significant side effects.

Data: mCD16−/−, mCD16−/− hCD16A+C57BI/6, mCD16−/− hCD16B+ and mCD16−/−hCD16A+hCD16B+mice from MacroGenics breeding colony were injected IP atdays 0, 2, 4, 7, 9, 11, 14, 16 and 18 with 2.4G2-3G8 DB (75 ug/mouse),or PBS. Blood was collected at days—10 (pre-bleed), 4, 11 and 18 forFACS analysis. Animal health and activity was recorded three times aweek.

Dose Blood Collection Group # of Animals μg/ms Test Article RouteTimepoints A 2mCD16−/− — PBS IP Days −10, 4, 11, 18 B 2mCD16−/− 16A+ B6— PBS IP Days −10, 4, 11, 18 C 2mCD16−/− 16B+ — PBS IP Days −10, 4, 11,18 D 2mCD16−/− 16A+ 16B+ — PBS IP Days −10, 4, 11, 18 E 6mCD16−/− 752.4G2-3G8 DB IP Days −10, 4, 11, 18 F 6mCD16−/− 16A+ B6 75 2.4G2-3G8 DBIP Days −10, 4, 11, 18 G 6mCD16−/− 16B+ 75 2.4G2-3G8 DB IP Days −10, 4,11, 18 H 6mCD16−/− 16A+ 16B+ 75 2.4G2-3G8 DB IP Days −10, 4, 11, 18

FACS analysis Method: Whole blood samples were collected 10 days priorto 2.4G2-3G8 administration and 4, 11 and 18 days after the initiationof the treatment. The blood samples were analyzed to determine theeffect of 2.4G2-3G8 on the B cell counts by a FACS based assay. Anon-wash protocol was used for B cell, T cell and PMN count by usingTruCOUNT tubes, obtained from BD Immunocytometry System. The panel ofantibodies used in the analysis was 1A8-FITC for PMN, CD3-PE for T cell,CD19-APC for B cell and CD45-PerCP for total leukocytes. Results

Mice treated with hE16 or 2.4G2-3G8 DB did not show any sign ofdiscomfort at anytime during the duration of the experimentation.

B cell depletion was observed in mCD16−/− hCD16A+ or mCD16−/−hCD16A+hCD16B+mice but not in mCD16−/− mice. These data indicate thathCD16A carrying effector cells were required for the B cell killing(FIG. 25). There were no significant changes for T cells and PMN levelduring the study.

Intravenous (IV) Model: The anti-tumor activity of MGD261 was testedusing an intravenous (IV) model of the human tumor cell line Raji. Rajiis a human Burkitt's lymphoma cell line expressing hCD32B. When injectedintravenously in mCD16−/−, hCD16A+, RAG1−/− mice, tumor cells locate tothe spine and results in hind leg paralysis.

Data indicate that MGD261 is capable of blocking Raji tumor cell growthin vivo in mCD16−/−, hCD16A+, RAG1−/− mice. Data indicate that MGD261can be used in the treatment of CD32B expressing B cell malignancies inthe human.

Data: Twelve-twenty week old mCD16−/−, hCD16A+, RAGI−/− C57BI/6 micefrom MacroGenics breeding colony were injected IV at day 0 with 5×10⁶Raji cells. At Days 6, 9, 13, 16, 20, 23, 27 and 30 mice were alsotreated intraperitoneously (IP) with 250, 25 or 2.5 ug MGD261 or withPBS (negative control). Mice were then observed daily and body weightwas recorded twice a week. Mice developing hind leg paralysis weresacrificed.

Results: Mice treated with PBS died between day 25 and day 50. Micetreated with MGD261 survived at least until day 90 (FIG. 26). Theincreased survival is statistically significant. A comparison ofsurvival curves using a Logrank Test gave a χ² of 96.46 (df 9; Pvalue<0.0001).

6.7 DART Expression in Prokaryotes

Experiments were conducted to demonstrate the ability to produce DARTsin non-mammalian hosts. Accordingly, Escherichia coli was transformedwith a DART-expressing plasmid, and DART expression was monitored.

Materials and Methods:

Plasmid construction: 3G8 is a humanized monoclonal antibody againstHuCD16. The DART described here consists of two covalently linkedchains, each of which has a VL followed by a spacer, then a VH followedby a Cys in a good context to form a disulfide bond to the oppositechain. The DART sequence encoding 3G8VL-GlyGlyGlySerGlyGlyGlyGly (SEQ IDNO: 10)-3G8VH-LeuGlyGlyCys was PCR amplified from an existing eukaryoticexpression construct and digested with Nco I and EcoR I. The targetvector was pET25b (+) (Novagen), which contains a pelB leader sequencefor secretion in E. coli. Prior to insertion of the 3G8/3G8 DARTsequences, the vector was modified as follows: First, the T7 promoterwas replaced by the lower activity lac promoter in order to favorsoluble, albeit lower level, expression of proteins under its control.Additionally, two point mutations were introduced to eliminate twointernal Met codons present at the beginning of the multiple cloningsite (MCS) in order to favor initiation at the Met present at thebeginning of the pelB leader. The DART that is produced by thisconstruct consists of two V-region arms that have the same specificity,namely HuCD16.

Expression: BL21 DE3 cells (Novagen) were transformed with the pET25b(+)T7-lac+3G8/3G8 plasmid and an amp-resistant colony was used to seedbroth culture. When the culture reached 0.5 OD600 units, 0.5 mM IPTG wasadded to induce expression. The culture was grown at 30° C. for 2 hoursand the cell-free medium was collected.

Purification: The 3G8/3G8 DART was purified in a two step processutilizing affinity and size exclusion chromatography. The DART wascaptured from the conditioned medium using affinity chromatography.Specifically, CD16A coupled to CNBr activated Sepharose 4B (GEHealthcare). The CD16A-Sepharose resin was equilibrated in 20 mMTris/HCl, pH 8.0 prior to loading. Upon completion of loading, the resinwas washed with equilibration buffer prior to elution of the bound DARTwith 50 mM Glycine pH 3.0. The eluted DART was immediately neutralizedwith 1M Tris/HCl pH 8.0 and concentrated using a centrifugation typeconcentrator (Vivaspin 20, 10k MWCO PES, VivaScience Inc.). Theconcentrated DART was further purified by size exclusion chromatographyusing a Superdex 200 column (GE Healthcare) equilibrated in PBS.

Results

1.7 liters of E. coli cultured conditioned medium was processed throughthe CD16A Sepharose column. The yield of DART was 0.12 mg. Analysis ofthe purified DART by SDS-PAGE and SEC demonstrated comparability to themammalian cell (CHO) expressed control DART (FIG. 27).

E. coli Expressed h3G8-h3G8 DART Binding ELISA: Expression of h3G8-h3G8DART in E. coli was measured using an ELISA. 50 μl/well of 2 μg/ml ofanti-h3G8 Fv specific antibody 2C11 was coated on 96-well Maxisorp platein Carbonate buffer at 4° C. over night. The plate was washed threetimes with PBS-T (PBS, 0.1% Tween 20) and then blocked by 0.5% BSA inPBS-T for 30 minutes at room temperature before adding testing DART.During blocking, E. coli expressed h3G8-h3G8 DART, h2B6-h3G8 DART, andh2B6-h2B6 DART (negative control) were diluted in 1 μg/ml, and 0.3 μg/mlin PBST/BSA. 50 μl/well of diluted DARTs were added to the each well.The plate was incubated at room temperature for 1 hour. After washingwith PBS-T three times, 50 μl/well of 0.1 μg/ml of Biotinlated sCD16-Fcfusion was added to the plate. The plate was incubated at roomtemperature for 1 hour. After washing with PBS-T three times, 50 μl/wellof a 1:5000 dilution of HRP conjugated streptavidin (Amersham PharmaciaBiotech) was used for detection and incubated at room temperature for 1hour. The plate was washed with PBS-T three times and developed using 80ul/well of TMB substrate. After 5 minutes incubation, the reaction wasstopped by 40 μl/well of 1% H₂SO₄. The OD450 nm was read by using a96-well plate reader and SOFTmax software. The read out was plottedusing GraphPadPrism 3.03 software (FIG. 28).

6.8 DART-Induced Human B-Cell Death

Human PBMC were incubated overnight with: CD16-CD32B hu3G8-hu2b6(described above); ch2B6-aglyc-aglycosylated chimeric 2B6 antibody(described in co-pending U.S. patent application Ser. No. 11/108,135,published as US2005/0260213, herein incorporated by reference) andCD16-CD79. The DNA and encoded protein sequences of CD16-CD79 are asfollows:

H3G8VL-CB3.1VH Nucleotide Sequence (SEQ ID NO: 226): gacatcgtgatgacccaatc tccagactct ttggctgtgt ctctagggga 50 gagggccacc atcaactgcaaggccagcca aagtgttgat tttgatggtg 100 atagttttat gaactggtac caacagaaaccaggacagcc acccaaactc 150 ctcatctata ctacatccaa tctagaatct ggggtcccagacaggtttag 200 tggcagtggg tctgggacag acttcaccct caccatcagc agcctgcagg250 ctgaggatgt ggcagtttat tactgtcagc aaagtaatga ggatccgtac 300acgttcggac aggggaccaa gcttgagatc aaaggaggcg gatccggagg 350 cggaggccaggtccaactgc agcagcctgg ggctgagctg gtgaggcctg 400 gggcttcagt gaagctgtcctgcaaggctt ctggctacac cttcaccagc 450 tactggatga actgggtgaa gcagaggcctggacaaggcc ttgaatggat 500 tggtatggtt gatccttcag acagtgaaac tcactacaatcaaatgttca 550 aggacaaggc cacattgact gttgacaaat cctccagcac agcctacatg600 cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag 650agctatgggc tactggggtc aaggaacctc agtcaccgtc tcctcagttg 700 agcccaaatcttgt 714 Amino Acid Sequence (SEQ ID NO: 227): DIVMTQSPDS LAVSLGERATINCKASQSVD FDGDSFMNWY QQKPGQPPKL 50 LIYTTSNLES GVPDRFSGSG SGTDFTLTISSLQAEDVAVY YCQQSNEDPY 100 TFGQGTKLEI KGGGSGGGGQ VQLQQPGAEL VRPGASVKLSCKASGYTFTS 150 YWMNWVKQRP GQGLEWIGMV DPSDSETHYN QMFKDKATLT VDKSSSTAYM200 QLSSLTSEDS AVYYCARAMG YWGQGTSVTV SSVEPKSC 238 CB3.1VL-h3G8VHNucleotide Sequence (SEQ ID NO: 228): gatgttgtga tgacccagac tccactcactttgtcggtta acattggaca 50 accagcctcc atctcttgta agtcaagtca gagcctcttagatactgatg 100 gaaagacata tttgaattgg ttgttacaga ggccaggcca gtctccaaac150 cgcctaatct atctggtgtc taaactggac tctggagtcc ctgacaggtt 200cactggcagt ggatcaggga cagatttcac actgaaaatc agcagagtgg 250 aggctgaggatttgggaatt tattattgct ggcaaggtac acattttccg 300 ctcacgttcg gtgctgggaccaagctggag ctgaaaggag gcggatccgg 350 aggcggaggc caggttaccc tgagagagtctggccctgcg ctggtgaagc 400 ccacacagac cctcacactg acttgtacct tctctgggttttcactgagc 450 acttctggta tgggtgtagg ctggattcgt cagcctcccg ggaaggctct500 agagtggctg gcacacattt ggtgggatga tgacaagcgc tataatccag 550ccctgaagag ccgactgaca atctccaagg atacctccaa aaaccaggta 600 gtcctcacaatgaccaacat ggaccctgtg gatactgcca catactactg 650 tgctcaaata aaccccgcctggtttgctta ctggggccaa gggactctgg 700 tcactgtgag ctcattcaac aggggagagt gt732 Amino Acid Sequence (SEQ ID NO: 229): DVVMTQTPLT LSVNIGQPASISCKSSQSLL DTDGKTYLNW LLQRPGQSPN 50 RLIYLVSKLD SGVPDRFTGS GSGTDFTLKISRVEAEDLGI YYCWQGTHFP 100 LTFGAGTKLE LKGGGSGGGG QVTLRESGPA LVKPTQTLTLTCTFSGFSLS 150 TSGMGVGWIR QPPGKALEWL AHIWWDDDKR YNPALKSRLT ISKDTSKNQV200 VLTMTNMDPV DTATYYCAQI NPAWFAYWGQ GTLVTVSSFN RGEC 244

Apoptosis was assayed by FACS analysis as the percentage ofPI+Annexin-V+population of B cells (CD20+ cells) on the total FSC/SSCungated population (FIG. 29).

6.9 8B5-CB3.1 DART

8B5VL-CB3.1VH-VEPKSC

8B5VL was amplified by using H9 and lgh630R as primers, ch8B5Lc astemplate. CB3.1VH was amplified by using lgh628F and lgh629R as primers,ch8B5Hc as template. The linker sequence was incorporated in the primerslgh630R and lgh628F. The c-terminal linker and stop codon wasincorporated in lgh629R primer. The PCR products were gel purified andmixed together in equal molar ratio, then amplified by using H9 andlgh629R as primers. The overlapped PCR product was then digested withNheI/EcoRI restriction endonucleases, and cloned into pCIneo vector.

CB3.1VL-8B5VH-FNRGEC

CB3.1VL was amplified by using H9 and lgh630R, which shared the samesequence as 8B5VL at FR4, as primers, and chCB3.1Lc as template. 8B5VHwas amplified by using lgh631F and lgh640R as primers, and ch8B5Hc astemplate. The linker sequence was incorporated in the primers lgh630Rand lgh631F. The c-terminal linker and stop codon was incorporated inlgh640R primer. The PCR products were gel purified and mixed together inequal molar ratio, then amplified by using H9 and lgh640R as primers.The overlapped PCR product was then digested with NheVEcoRI restrictionendonucleases, and cloned into pCIneo vector.

Anti-Flag tag-8B5VL-CB3.1VH-VEPKSC

Anti-Flag tag was inserted between signal sequence and 8B5VL byoverlapping PCR. The signal sequence and Flag tag was amplified by usingH9 and lgh647R as primers and ch8B5Lc as temperate. 8B5VL-CB3.1VH-VEPKSCwas re-amplified by using lgh647F and lgh629R as primers and8B5VL-CB3.1VH-VEPKSC as temperate. The PCR products were gel purifiedand mixed together in equal molar ratio, then amplified by using H9 andlgh629R as primers. The overlapped PCR product was then digested withNheI/EcoRI restriction endonucleases, and cloned into pCIneo vector.

8B5VL-CB3.1VH-LGGC

To generate a different C-terminal linker in 8B5VL-CB3.1VH-VEPKSCconstruct, the construct was re-amplified by using H9 and lgh646R asprimers. The C-terminal LGGC linker was integrated in lgh646R primer.The PCR product was then digested with NheI/EcoRI restrictionendonucleases, and cloned into pCIneo vector.

CB3.1VL-8B5VH-LGGC

The same strategy was used to create CB3.1VL-8B5VH-LGGC. The C-terminalLGGC linker was integrated in lgh648R primer and CB3.1VL-8B5VH-FNRGECwas used as temperate. The PCR product was then digested with NheI/EcoRIrestriction endonucleases, and cloned into pCIneo vector.

Anti-Flag tag-8B5VL-CB3.1VH-LGGC

The same strategy was also used to create Anti-Flagtag-8B5VL-CB3.1VH-LGGC. The C-terminal LGGC linker was integrated inlgh648R primer and Anti-Flag tag-8B5VL-CB3.1VH-VEPKSC was used astemperate. The PCR product was then digested with NheI/EcoRI restrictionendonucleases, and cloned into pCIneo vector.

8B5-CB3.1-VEPKSC Nucleotide sequence (SEQ ID NO: 230): gacattcagatgacacagtc tccatcctcc ctacttgcgg cgctgggaga 50 aagagtcagt ctcacttgtcgggcaagtca ggaaattagt ggttacttaa 100 gctggcttca gcagaaacca gatggaactattaaacgcct gatctacgcc 150 gcatccactt tagattctgg tgtcccaaaa aggttcagtggcagtgagtc 200 tgggtcagat tattctctca ccatcagcag tcttgagtct gaagattttg250 cagactatta ctgtctacaa tattttagtt atccgctcac gttcggtgct 300gggaccaagc tggagctgaa aggaggcgga tccggaggcg gaggccaggt 350 ccaactgcagcagcctgggg ctgagctggt gaggcctggg gcttcagtga 400 agctgtcctg caaggcttctggctacacct tcaccagcta ctggatgaac 450 tgggtgaagc agaggcctgg acaaggccttgaatggattg gtatggttga 500 tccttcagac agtgaaactc actacaatca aatgttcaaggacaaggcca 550 cattgactgt tgacaaatcc tccagcacag cctacatgca gctcagcagc600 ctgacatctg aggactctgc ggtctattac tgtgcaagag ctatgggcta 650ctggggtcaa ggaacctcag tcaccgtctc ctcagttgag cccaaatctt 700 gt 7028B5-CB3.1-VEPKSC Amino acid sequence (SEQ ID NO: 231): DIQMTQSPSSLLAALGERVS LTCRASQEIS GYLSWLQQKP DGTIKRLIYA 50 ASTLDSGVPK RFSGSESGSDYSLTISSLES EDFADYYCLQ YFSYPLTFGA 100 GTKLELKGGG SGGGGQVQLQ QPGAELVRPGASVKLSCKAS GYTFTSYWMN 150 WVKQRPGQGL EWIGMVDPSD SETHYNQMFK DKATLTVDKSSSTAYMQLSS 200 LTSEDSAVYY CARAMGYWGQ GTSVTVSSVE PKSC 234CB3.1-8B5-FNRGEC Nucleotide sequence (SEQ ID NO: 232): gatgttgtgatgacccagac tccactcact ttgtcggtta acattggaca 50 accagcctcc atctcttgtaagtcaagtca gagcctctta gatactgatg 100 gaaagacata tttgaattgg ttgttacagaggccaggcca gtctccaaac 150 cgcctaatct atctggtgtc taaactggac tctggagtccctgacaggtt 200 cactggcagt ggatcaggga cagatttcac actgaaaatc agcagagtgg250 aggctgagga tttgggaatt tattattgct ggcaaggtac acattttccg 300ctcacgttcg gtgctgggac caagctggag ctgaaaggag gcggatccgg 350 aggcggaggcgaagtgaagc ttgaggagtc tggaggaggc ttggtgcaac 400 ctggaggatc catgaaactctcttgtgaag cctctggatt cacttttagt 450 gacgcctgga tggactgggt ccgtcagtctccagagaagg ggcttgagtg 500 ggttgctgaa attagaaaca aagctaaaaa tcatgcaacatactatgctg 550 agtctgtgat agggaggttc accatctcaa gagatgattc caaaagtagt600 gtctacctgc aaatgaacag cttaagagct gaagacactg gcatttatta 650ctgtggggct ctgggccttg actactgggg ccaaggcacc actctcacag 700 tctcctcgttcaacagggga gagtgt 726 CB3.1-8B5-FNRGEC Amino acid sequence (SEQ ID NO:233): DVVMTQTPLT LSVNIGQPAS ISCKSSQSLL DTDGKTYLNW LLQRPGQSPN 50RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGI YYCWQGTHFP 100 LTFGAGTKLELKGGGSGGGG EVKLEESGGG LVQPGGSMKL SCEASGFTFS 150 DAWMDWVRQS PEKGLEWVAEIRNKAKNHAT YYAESVIGRF TISRDDSKSS 200 VYLQMNSLRA EDTGIYYCGA LGLDYWGQGTTLTVSSFNRG EC 242

8B5VL-CB3.1VH-LGGC

8B5VL was amplified by using H9 and lgh694R as primers, ch8B5Lc astemplate. 8B5VH was amplified by using lgh695F and lgh696R as primers,ch8B5Hc as template. The linker sequence was incorporated in the primerslgh694R and lgh695F. HuIgG1Fc was amplified by using lgh355F and lgh366Ras primers, ch8B5Hc as template. The PCR products were gel purified andmixed together in equal molar ratio, then amplified by using H9 andlgh366R as primers. The overlapped PCR product was then digested withNheI/EcoRI restriction endonucleases, and cloned into pCIneo vector.

8B5VL-CB3.1VH-LGGC Nucleotide sequence (SEQ ID NO: 234): gacattcagatgacacagtc tccatcctcc ctacttgcgg cgctgggaga 50 aagagtcagt ctcacttgtcgggcaagtca ggaaattagt ggttacttaa 100 gctggcttca gcagaaacca gatggaactattaaacgcct gatctacgcc 150 gcatccactt tagattctgg tgtcccaaaa aggttcagtggcagtgagtc 200 tgggtcagat tattctctca ccatcagcag tcttgagtct gaagattttg250 cagactatta ctgtctacaa tattttagtt atccgctcac gttcggtgct 300gggaccaagc tggagctgaa aggaggcgga tccggaggcg gaggccaggt 350 ccaactgcagcagcctgggg ctgagctggt gaggcctggg gcttcagtga 400 agctgtcctg caaggcttctggctacacct tcaccagcta ctggatgaac 450 tgggtgaagc agaggcctgg acaaggccttgaatggattg gtatggttga 500 tccttcagac agtgaaactc actacaatca aatgttcaaggacaaggcca 550 cattgactgt tgacaaatcc tccagcacag cctacatgca gctcagcagc600 ctgacatctg aggactctgc ggtctattac tgtgcaagag ctatgggcta 650ctggggtcaa ggaacctcag tcaccgtctc ctcactggga ggctgc 6968B5VL-CB3.1VH-LGGC Amino acid sequence (SEQ ID NO: 235): DIQMTQSPSSLLAALGERVS LTCRASQEIS GYLSWLQQKP DGTIKRLIYA 50 ASTLDSGVPK RFSGSESGSDYSLTISSLES EDFADYYCLQ YFSYPLTFGA 100 GTKLELKGGG SGGGGQVQLQ QPGAELVRPGASVKLSCKAS GYTFTSYWMN 150 WVKQRPGQGL EWIGMVDPSD SETHYNQMFK DKATLTVDKSSSTAYMQLSS 200 LTSEDSAVYY CARAMGYWGQ GTSVTVSSLG GC 232 CB3.1-8B5-LGGCNucleotide sequence (SEQ ID NO: 236): gatgttgtga tgacccagac tccactcactttgtcggtta acattggaca 50 accagcctcc atctcttgta agtcaagtca gagcctcttagatactgatg 100 gaaagacata tttgaattgg ttgttacaga ggccaggcca gtctccaaac150 cgcctaatct atctggtgtc taaactggac tctggagtcc ctgacaggtt 200cactggcagt ggatcaggga cagatttcac actgaaaatc agcagagtgg 250 aggctgaggatttgggaatt tattattgct ggcaaggtac acattttccg 300 ctcacgttcg gtgctgggaccaagctggag ctgaaaggag gcggatccgg 350 aggcggaggc gaagtgaagc ttgaggagtctggaggaggc ttggtgcaac 400 ctggaggatc catgaaactc tcttgtgaag cctctggattcacttttagt 450 gacgcctgga tggactgggt ccgtcagtct ccagagaagg ggcttgagtg500 ggttgctgaa attagaaaca aagctaaaaa tcatgcaaca tactatgctg 550agtctgtgat agggaggttc accatctcaa gagatgattc caaaagtagt 600 gtctacctgcaaatgaacag cttaagagct gaagacactg gcatttatta 650 ctgtggggct ctgggccttgactactgggg ccaaggcacc actctcacag 700 tctcctcgct gggaggctgc 720CB3.1-8B5-LGGC Amino acid sequence (SEQ ID NO: 237): DVVMTQTPLTLSVNIGQPAS ISCKSSQSLL DTDGKTYLNW LLQRPGQSPN 50 RLIYLVSKLD SGVPDRFTGSGSGTDFTLKI SRVEAEDLGI YYCWQGTHFP 100 LTFGAGTKLE LKGGGSGGGG EVKLEESGGGLVQPGGSMKL SCEASGFTFS 150 DAWMDWVRQS PEKGLEWVAE IRNKAKNHAT YYAESVIGRFTISRDDSKSS 200 VYLQMNSLRA EDTGIYYCGA LGLDYWGQGT TLTVSSLGGC 240 Primers:Lgh628F (SEQ ID NO: 238): ggaggcggat ccggaggcgg aggccaggtc caactgcagcagcctgg 47 Lgh629R (SEQ ID NO: 239): tttgaattct aacaagattt gggctcaactgaggagacgg tgactgagg 49 Lgh630R (SEQ ID NO: 240): gcctccgcct ccggatccgcctcctttcag ctccagcttg gtccc 45 Lgh631F (SEQ ID NO: 241): ggaggcggatccggaggcgg aggcgaagtg aagcttgagg agtctgg 47 Lgh640R (SEQ ID NO: 242):tttgaattct aacactctcc cctgttgaac gaggagactg tgagagtgg 49 Lgh644R (SEQ IDNO: 243): tttgtcgtca tcatcgtctt tgtagtcgga gtggacacct gtggagag 48Lgh646R (SEQ ID NO: 244): tttgaattct agcagcctcc cagtgaggag acggtgactg ag42 Lgh647F (SEQ ID NO: 245): caaagacgat gatgacgaca aagacattca gatgacacagtctcc 45 Lgh648R (SEQ ID NO: 246): tttgaattct agcagcctcc cagcgaggagactgtgagag tgg 43

Expression: The construct 5 and 6, or 6 and 7, or 8 and 9, or 9 and 10,encoded expression plasmids (FIG. 30) were co-transfected into HEK-293cells to express 8B5-CB3.1 DART with or without anti flag tag usingLipofectamine 2000 (Invitrogen). The conditioned medium was harvested inevery three days for three times. The conditioned medium was thenpurified using CD32B affinity column.

ELISA: ELISA were conducted as follows: 50 μl/well of 2 ug/ml ofCD32B-Fc was coated on 96-well Maxisorp plate in Carbonate buffer at 4°C. over night. The plate was washed three times with PBS-T (PBS, 0.1%Tween 20) and then blocked by 0.5% BSA in PBS-T for 30 minutes at roomtemperature before adding testing single chain Fc fusion protein. Duringblocking, 8B5-CB3.1 DART was diluted in a serial of two-fold dilutionstarting at 2 μg/ml. 25 μl/well of diluted DART mixed with 25 μl/well of50 ng/ml ch8B5 was transferred from dilution plate to the ELISA plate.The plate was incubated at room temperature for 1 hour. After washingwith PBS-T three times, 50 μl/well of 1:10,000 diluted HRP conjugatedF(ab′)₂ goat anti human IgG F(ab′)₂ (Jackson ImmunoResearch) was addedto the plate. The plate was incubated at room temperature for 1 hour.The plate was washed with PBS-T three times and developed with 80μl/well of TMB substrate. After 5 minutes incubation, the reaction wasstopped by 40 μl/well of 1% H₂SO₄. The OD450 nm was read using a 96-wellplate reader and SOFTmax software. The read out was plotted usingGraphPadPrism 3.03 software (FIG. 31).

6.10 Design and Characterization of Ig-Like Tetravalent DART

Four polypeptide chains were employed to produce an Ig-like DART specieshaving tetravalent antigen binding sites (FIG. 32; FIG. 33). The Ig-likeDART species has unique properties, since its domains may be designed tobind to the same epitope (so as to form a tetravalent, mono-epitopespecific Ig-like DART capable of binding four identical antigenmolecules), or to different epitopes or antigens For example, itsdomains may be designed to bind to two epitopes of the same antigen (soas to form a tetravalent, mono-antigen specific, bi-epitope specificIg-like DART), or to epitopes of different antigen molecules so as toform a tetravalent Ig-like DART having a pair of binding sites specificfor a first antigen and a second pair of binding sites specific for asecond antigen). Hybrid molecules having combinations of such attributescan be readily produced.

To illustrate the characteristics of such Ig-like DART species, anexemplary tetravalent Ig-like DART species was produced having a pair ofbinding sites specific for CD32 and a second pair of binding sitesspecific CD16A. This Ig-like DART species was produced using thefollowing four polypeptide chains:

2.4G2-3G8-hKappa Nucleotide Sequence (SEQ ID NO: 247): gatgtccagatgacccagtc tccatctaat cttgctgcct ctcctggaga 50 aagtgtttcc atcaattgcaaggcaagtga gagcattagc aagtatttag 100 cctggtatct acagaaacct gggaaagcaaataagcttct tatgtacgat 150 gggtcaactt tgcaatctgg aattccatcg aggttcagtggcagtggatc 200 tggtacagat ttcactctca ccatcagaag cctggagcct gaagattttg250 gactctatta ctgtcaacag cattatgaat atccagccac gttcggttct 300gggaccaagc tggagatcaa aggaggcgga tccggaggcg gaggccaggt 350 taccctgaaagagtctggcc ctgggatatt gcagccctcc cagaccctca 400 gtctgacttg ttctttctctgggttttcac tgaggacttc tggtatgggt 450 gtaggctgga ttcgtcagcc ttcagggaagggtctagagt ggctggcaca 500 catttggtgg gatgatgaca agcgctataa tccagccctgaagagccgac 550 tgacaatctc caaggatacc tccagcaacc aggtattcct caaaatcgcc600 agtgtggaca ctgcagatac tgccacatac tactgtgctc aaataaaccc 650cgcctggttt gcttactggg gccaagggac tctggtcact gtgagctcac 700 tgggaggctgcggcggaggg agccgtacgg tggctgcacc atcggtcttc 750 atcttcccgc catctgatgagcagttgaaa tctggaactg cctctgttgt 800 gtgcctgctg aataacttct atcccagagaggccaaagta cagtggaagg 850 tggataacgc cctccaatcg ggtaactccc aggagagtgtcacagagcag 900 gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa950 agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg 1000gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 1044 2.4G2-3G8-hKappaEncoded Amino Acid Sequence (SEQ ID NO: 248): DVQMTQSPSN LAASPGESVSINCKASESIS KYLAWYLQKP GKANKLLMYD 50 GSTLQSGIPS RFSGSGSGTD FTLTIRSLEPEDFGLYYCQQ HYEYPATFGS 100 GTKLEIKGGG SGGGGQVTLK ESGPGILQPS QTLSLTCSFSGFSLRTSGMG 150 VGWIRQPSGK GLEWLAHIWW DDDKRYNPAL KSRLTISKDT SSNQVFLKIA200 SVDTADTATY YCAQINPAWF AYWGQGTLVT VSSLGGCGGG SRTVAAPSVF 250IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ 300 DSKDSTYSLSSTLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 350 3G8-2.4G2-hG1 NucleotideSequence (SEQ ID NO: 249): gacactgtgc tgacccaatc tccagcttct ttggctgtgtctctagggca 50 gagggccacc atctcctgca aggccagcca aagtgttgat tttgatggtg 100atagttttat gaactggtac caacagaaac caggacagcc acccaaactc 150 ctcatctatactacatccaa tctagaatct gggatcccag ccaggtttag 200 tgccagtggg tctgggacagacttcaccct caacatccat cctgtggagg 250 aggaggatac tgcaacctat tactgtcagcaaagtaatga ggatccgtac 300 acgttcggag gggggaccaa gctggaaata aaaggaggcggatccggagg 350 cggaggcgag gtggagctag tggagtctgg gggaggctta gtgcagcctg400 gaaggtccct gaaactctcg tgtgcagcct caggattcac tttcagtgac 450tattacatgg cctgggtccg gcaggctcca acgacgggtc tggagtgggt 500 cgcatccattagttatgatg gtggtgacac tcactatcga gactccgtga 550 agggccgatt tactatttccagagataatg caaaaagcag cctatacctg 600 caaatggaca gtctgaggtc tgaggacacggccacttatt actgtgcaac 650 agagactacg ggaataccta caggtgttat ggatgcctggggtcaaggag 700 tttcagtcac tgtctcctca ctgggaggct gcggcggagg gagcgcctcc750 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc 800tgggggcaca gcggccctgg gctgcctggt caaggactac ttccccgaac 850 cggtgacggtgtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 900 ttcccggctg tcctacagtcctcaggactc tactccctca gcagcgtggt 950 gaccgtgccc tccagcagct tgggcacccagacctacatc tgcaacgtga 1000 atcacaagcc cagcaacacc aaggtggaca agagagttgagcccaaatct 1050 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg1100 gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 1150tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1200 gaccctgaggtcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa 1250 tgccaagaca aagccgcgggaggagcagta caacagcacg taccgtgtgg 1300 tcagcgtcct caccgtcctg caccaggactggctgaatgg caaggagtac 1350 aagtgcaagg tctccaacaa agccctccca gcccccatcgagaaaaccat 1400 ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc1450 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1500aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 1550 gccggagaacaactacaaga ccacgcctcc cgtgctggac tccgacggct 1600 ccttcttcct ctacagcaagctcaccgtgg acaagagcag gtggcagcag 1650 gggaacgtct tctcatgctc cgtgatgcatgaggctctgc acaaccacta 1700 cacgcagaag agcctctccc tgtctccggg taaa 17343G8-2.4G2-hG1 Encoded Amino Acid Sequence (SEQ ID NO: 250): DTVLTQSPASLAVSLGQRAT ISCKASQSVD FDGDSFMNWY QQKPGQPPKL 50 LIYTTSNLES GIPARFSASGSGTDFTLNIH PVEEEDTATY YCQQSNEDPY 100 TFGGGTKLEI KGGGSGGGGE VELVESGGGLVQPGRSLKLS CAASGFTFSD 150 YYMAWVRQAP TTGLEWVASI SYDGGDTHYR DSVKGRFTISRDNAKSSLYL 200 QMDSLRSEDT ATYYCATETT GIPTGVMDAW GQGVSVTVSS LGGCGGGSAS250 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT 300FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS 350 CDKTHTCPPCPAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 400 DPEVKFNWYV DGVEVHNAKTKPREEQYNST YRVVSVLTVL HQDWLNGKEY 450 KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV 500 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSKLTVDKSRWQQ 550 GNVFSCSVMH EALHNHYTQK SLSLSPGK 578

Preparations of Ig-like DART molecules having the above sequences wereobtained from different plasmid isolates and were denominated “Ig DART1” and “Ig DART 2.” The ability of these Ig-like DART species to bindmCD32-hCD16A in an ELISA was compared with that of medium alone, a DARThaving a single CD32 and a single CD16A binding site (“DART”), andcontrol anti-ch-mCD32 mAb (FIG. 34). The Ig-like DART of the presentinvention was found to have much greater antigen binding affinity thaneither DART or the control antibody.

6.11 Design and Characterization of CD32B-CD79-1 and CD32B-CD79-2Bispecific Diabodies

Genes encoding CD79VL-CD32BVH (Sequence 1), CD32BVL-CD79VH-1 (Sequence2), and CD32BVL-CD79VH-2 (Sequence 3) were cloned into expression vectorpEE13 resulting in expression constructs 1, 2, and 3 respectively. Theconstruct 1 expression plasmid was co-transfected together with eitherexpression plasmid 2 or 3 into HEK-293 cells to make CD32B-CD79-1 andCD32B-CD79-2 bispecific diabodies, respectively. The conditioned mediumwas harvested in every three days for three times. The conditionedmedium was then purified using CD32B affinity column.

ELISA were conducted as follows: 50 μl/well of 2 μg/ml of CD32B-Fc wascoated on 96-well Maxisorp plate in Carbonate buffer at 4° C. overnight. The plate was washed three times with PBS-T (PBS, 0.1% Tween 20)and then blocked by 0.5% BSA in PBS-T for 30 minutes at room temperaturebefore adding testing single chain Fc fusion protein. During blocking,the CD32B-CD79-1 or CD32B-CD79-2 bispecific diabody was diluted in aserial of two-fold dilution starting at 2 μg/ml. 25 μl/well of dilutedbispecific diabody was mixed with 25 μl/well of 50 ng/ml anti-CD32Bantibody and added to an ELISA plate. The plate was incubated at roomtemperature for 1 hour. After washing with PBS-T three times, 50 μl/wellof 1:10,000 diluted HRP conjugated F(ab′)₂ goat anti human IgG F(ab′)₂(Jackson ImmunoResearch) was added to the plate. The plate was incubatedat room temperature for 1 hour. The plate was washed with PBS-T threetimes and developed with 80 μl/well of TMB substrate. After 5 minutesincubation, the reaction was stopped by 40 μl/well of 1% H₂SO₄. TheOD450 nm was read using a 96-well plate reader and SOFTmax software. Theread out was plotted using GraphPadPrism 3.03 software. The experimentrevealed that the CD32B-CD79-1 and CD32B-CD79-2 bispecific Diabodieswere capable of immunospecific binding to CD32-Fc with an affinityequivalent to that of the anti-CD32B control antibody. The nucleotideand encoded amino acid sequences of the above-described constructs areprovided below:

Sequence 1 - CD79VL-CD32BVH nucleotide sequence (SEQ ID NO: 251):gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca 50 gccggcctccatctcctgca agtcaagtca gagcctctta gatagtgatg 100 gaaagacata tttgaattggtttcagcaga ggccaggcca atctccaaac 150 cgcctaattt atctggtgtc taaactggactctggggtcc cagacagatt 200 cagcggcagt gggtcaggca ctgatttcac actgaaaatcagcagggtgg 250 aggctgagga tgttggggtt tattactgct ggcaaggtac acattttccg300 ctcacgttcg gcggagggac caagcttgag atcaaaggag gcggatccgg 350aggcggaggc gaagtgaagc ttgaggagtc tggaggaggc ttggtgcaac 400 ctggaggatccatgaaactc tcttgtgaag cctctggatt cacttttagt 450 gacgcctgga tggactgggtccgtcagtct ccagagaagg ggcttgagtg 500 ggttgctgaa attagaaaca aagctaaaaatcatgcaaca tactatgctg 550 agtctgtgat agggaggttc accatctcaa gagatgattccaaaagtagt 600 gtctacctgc aaatgaacag cttaagagct gaagacactg gcatttatta650 ctgtggggct ctgggccttg actactgggg ccaaggcacc actctcacag 700tctcctcgct gggaggctgc 720 Sequence 2 - CD79VL-CD32BVH amino acidesequence (SEQ ID NO: 252): DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTYLNWFQQRPGQSPN 50 RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP 100LTFGGGTKLE IKGGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS 150 DAWMDWVRQAPGKGLEWVAE IRNKAKNHAT YYAESVIGRF TISRDDAKNS 200 LYLQMNSLRA EDTAVYYCGALGLDYWGQGT LVTVSSLGGC 240 Sequence 3 - CD32BVL-CD79VH-1 nucleotidesequence (SEQ ID NO: 253): gacatccaga tgacccagtc tccatcctcc ttatctgcctctgtgggaga 50 tagagtcacc atcacttgtc gggcaagtca ggaaattagt ggttacttaa 100gctggctgca gcagaaacca ggcaaggccc ctagacgcct gatctacgcc 150 gcatccactttagattctgg tgtcccatcc aggttcagtg gcagtgagtc 200 tgggaccgag ttcaccctcaccatcagcag ccttcagcct gaagattttg 250 caacctatta ctgtctacaa tattttagttatccgctcac gttcggaggg 300 gggaccaagg tggaaataaa aggaggcgga tccggaggcggaggccaggt 350 tcagctggtg cagtctggag ctgaggtgaa gaagcctggc gcctcagtga400 aggtctcctg caaggcttct ggttacacct ttaccagcta ctggatgaac 450tgggtgcgac aggcccctgg acaagggctt gagtggatcg gaatgattga 500 tccttcagacagtgaaactc actacaatca aatgttcaag gacagagtca 550 ccatgaccac agacacatccacgagcacag cctacatgga gctgaggagc 600 ctgagatctg acgacacggc cgtgtattactgtgcgagag ctatgggcta 650 ctgggggcaa gggaccacgg tcaccgtctc ctcactgggaggctgc 696 Sequence 4 - CD32BVL-CD79VH-1 amino acid sequence (SEQ ID NO:254): DIQMTQSPSS LSASVGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA 50ASTLDSGVPS RFSGSESGTE FTLTISSLQP EDFATYYCLQ YFSYPLTFGG 100 GTKVEIKGGGSGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYTFTSYWMN 150 WVRQAPGQGL EWIGMIDPSDSETHYNQMFK DRVTMTTDTS TSTAYMELRS 200 LRSDDTAVYY CARAMGYWGQ GTTVTVSSLG GC232 Sequence 5 - CD32BVL-CD79VH-2 nucleotide sequence (SEQ ID NO: 255):gacatccaga tgacccagtc tccatcctcc ttatctgcct ctgtgggaga 50 tagagtcaccatcacttgtc gggcaagtca ggaaattagt ggttacttaa 100 gctggctgca gcagaaaccaggcaaggccc ctagacgcct gatctacgcc 150 gcatccactt tagattctgg tgtcccatccaggttcagtg gcagtgagtc 200 tgggaccgag ttcaccctca ccatcagcag ccttcagcctgaagattttg 250 caacctatta ctgtctacaa tattttagtt atccgctcac gttcggaggg300 gggaccaagg tggaaataaa aggaggcgga tccggaggcg gaggccaggt 350tcagctggtg cagtctggag ctgaggtgaa gaagcctggc gcctcagtga 400 aggtctcctgcaaggcttct ggttacacct ttaccagcta ctggatgaac 450 tgggtgcgac aggcccctggacaagggctt gagtggatcg gaatgattga 500 tccttcagac agtgaaactc actacaatcaaaagttcaag gacagagtca 550 ccatgaccac agacacatcc acgagcacag cctacatggagctgaggagc 600 ctgagatctg acgacacggc cgtgtattac tgtgcgagag ctatgggcta650 ctgggggcaa gggaccacgg tcaccgtctc ctcactggga ggctgc 696 Sequence 6 -CD32BVL-CD79VH-2 amino acid sequence (SEQ ID NO: 256): DIQMTQSPSSLSASVGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA 50 ASTLDSGVPS RFSGSESGTEFTLTISSLQP EDFATYYCLQ YFSYPLTFGG 100 GTKVEIKGGG SGGGGQVQLV QSGAEVKKPGASVKVSCKAS GYTFTSYWMN 150 WVRQAPGQGL EWIGMIDPSD SETHYNQKFK DRVTMTTDTSTSTAYMELRS 200 LRSDDTAVYY CARAMGYWGQ GTTVTVSSLG GC 232

6.12 Construction and Optimization of H8B5-HBCRC Bio-FunctionalDiabodies

A diabody was constructed that contains variable regions capable ofbinding to CD32 and B-cell receptor complex (“BCRC”).

Cloning. The constructs were constructed using standard PCR/overlappingPCR:

h8B5VL-G3SG4-hBCRCVH M481-LGGC:

-   -   A fully humanized 8B5 VL (recognizing CD32) was amplified by        using lgh321F and lgh788R as primers. hBCRCVH M48I was amplified        by using lgh784F and lgh386R as primers. The PCR products were        gel purified and mix together and amplified by using lgh321F and        lgh386R. The overlapping PCR fragment was then cloned into pEE6        at XbaI-EcoRI site. “G3SG4” is a linker having the sequence:        GGGSGGGG (SEQ ID NO: 10).        hBCRCVL R45N-G3SG4-h8B5VH LGGC    -   The hBCRCVL R45N was amplified by using lgh321F and lgh785R as        primers. The h8B5VH was amplified by using lgh787F and lgh786R        as primers. The PCR products were gel purified and mix together        and amplified by using lgh321F and lgh786R. The overlapping PCR        fragment was then cloned into pEE13 at XbaI-EcoRI site.

Single Vector Construction. The pEE6hHBCRCVL R45N-h8B5VH was digested atBgl II-Sal I sites and a 3.3 kb fragment was purified and inserted intopEE13 hHBCRCVL 45N-h8B5VH at BamHI-SalI sites. The Bgl II and BamH Ishares compatible cohesive ends. The sequence of the DART and primersused for constructing the DART are depicted below:

hHBCRCVL. R45N-h8B5VH-LGGC nucleotide sequence (SEQ ID NO: 257):gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca 50 gccggcctccatctcctgca agtcaagtca gagcctctta gatagtgatg 100 gaaagacata tttgaattggtttcagcaga ggccaggcca atctccaaac 150 cgcctaattt atctggtgtc taaactggactctggggtcc cagacagatt 200 cagcggcagt gggtcaggca ctgatttcac actgaaaatcagcagggtgg 250 aggctgagga tgttggggtt tattactgct ggcaaggtac acattttccg300 ctcacgttcg gcggagggac caagcttgag atcaaaggag gcggatccgg 350aggcggaggc gaagtgaagc ttgaggagtc tggaggaggc ttggtgcaac 400 ctggaggatccatgaaactc tcttgtgaag cctctggatt cacttttagt 450 gacgcctgga tggactgggtccgtcagtct ccagagaagg ggcttgagtg 500 ggttgctgaa attagaaaca aagctaaaaatcatgcaaca tactatgctg 550 agtctgtgat agggaggttc accatctcaa gagatgattccaaaagtagt 600 gtctacctgc aaatgaacag cttaagagct gaagacactg gcatttatta650 ctgtggggct ctgggccttg actactgggg ccaaggcacc actctcacag 700tctcctcgct gggaggctgc 720 hHBCRCVL. R45N-h8B5VH-LGGC amino acidesequence (SEQ ID NO: 258): DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTYLNWFQQRPGQSPN 50 RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP 100LTFGGGTKLE IKGGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFS 150 DAWMDWVRQAPGKGLEWVAE IRNKAKNHAT YYAESVIGRF TISRDDAKNS 200 LYLQMNSLRA EDTAVYYCGALGLDYWGQGT LVTVSSLGGC 240 H8B5VL-hHBCRCVH M48I-LGGC nucleotide sequence(SEQ ID NO: 259): gacatccaga tgacccagtc tccatcctcc ttatctgcct ctgtgggaga50 tagagtcacc atcacttgtc gggcaagtca ggaaattagt ggttacttaa 100 gctggctgcagcagaaacca ggcaaggccc ctagacgcct gatctacgcc 150 gcatccactt tagattctggtgtcccatcc aggttcagtg gcagtgagtc 200 tgggaccgag ttcaccctca ccatcagcagccttcagcct gaagattttg 250 caacctatta ctgtctacaa tattttagtt atccgctcacgttcggaggg 300 gggaccaagg tggaaataaa aggaggcgga tccggaggcg gaggccaggt350 tcagctggtg cagtctggag ctgaggtgaa gaagcctggc gcctcagtga 400aggtctcctg caaggcttct ggttacacct ttaccagcta ctggatgaac 450 tgggtgcgacaggcccctgg acaagggctt gagtggatcg gaatgattga 500 tccttcagac agtgaaactcactacaatca aatgttcaag gacagagtca 550 ccatgaccac agacacatcc acgagcacagcctacatgga gctgaggagc 600 ctgagatctg acgacacggc cgtgtattac tgtgcgagagctatgggcta 650 ctgggggcaa gggaccacgg tcaccgtctc ctcactggga ggctgc 696H8B5VL-HBCRCVH M48I-LGGC amino acid sequence (SEQ ID NO: 260):DIQMTQSPSS LSASVGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA 50 ASTLDSGVPSRFSGSESGTE FTLTISSLQP EDFATYYCLQ YFSYPLTFGG 100 GTKVEIKGGG SGGGGQVQLVQSGAEVKKPG ASVKVSCKAS GYTFTSYWMN 150 WVRQAPGQGL EWIGMIDPSD SETHYNQMFKDRVTMTTDTS TSTAYMELRS 200 LRSDDTAVYY CARAMGYWGQ GTTVTVSSLG GC 232Lgh321F Primer (SEQ ID NO: 261): cgagctagct ctagatgaga tcacagttct ctctac36 Lgh386R Primer (SEQ ID NO: 262): tttgaattct agcagcctcc cagtgaggagacggtgaccg tggtc 45 Lgh784F Primer (SEQ ID NO: 263): ggcggatccggaggcggagg ccaggttcag ctggtgcag 39 Lgh785R Primer (SEQ ID NO: 264):cctccggatc cgcctccttt gatctcaagc ttggtccc 38 Lgh786R Primer (SEQ ID NO:265): tttgaattct agcagcctcc caggctggag acggtcacca gg 42 Lgh787F Primer(SEQ ID NO: 266): ggaggcggat ccggaggcgg aggcgaagtg cagcttgtgg agtc 44

The Hu3G8VL 1-G3SG4-Hu2B6VH 4-LGGC expression plasmid was co-transfectedtogether with Hu2B6VL 5-G3SG4-Hu3G8VH 5-LGGC into HEK-293 cells to makeHu2B6 4.5-Hu3G8 5.1 biospecific diabody recognizing CD32 and CD79. Atthe same time, Hu2B6VL 5-G3SG4-Hu2B6VH 4-LGGC and Hu3G8VL1-G3SG4-Hu3G8VH 5-LGGC were transfected individually into HEK-293 cellsto make Hu2B6 4.5 and Hu3G8 5.1 diabody. After three days in culture,the conditioned medium were harvested and characterized by bindingELISA. The result of this experiment is depicted in FIG. 36.

Experimental Design: 100 ng/well of soluble FcRIIb-G2-Agly was coated on96-well Maxisorp plate in Carbonate buffer at 40° C. overnight. Platewas washed three times with PBS/0.1% Tween20 and then blocked by 0.5%BSA in PBS/0.1% Tween 20 for 30 mins at room temperature before addingdiabodies. A serial of two-fold dilution of conditioned medium of Hu2B64.5-Hu3G8 5.1 biospecific diabody, Hu2B6 4.5 diabody, and hu3G8 5.1diabody starting from 25 ng/well was added to the each well. The platewas incubated at room temperature for 1 hour. After washed with PBS/0.1%Tween20 three times, 10 ng/well of FcRIIIa-G2-Biotin was added to theplate. The plate was incubated at room temperature for 1 hour. Afterwashed with PBS/0.1% Tween20 three times, 50 ul of 1:5000 dilution ofHRP conjugated Streptavidin (Amersham Pharmacia Biotech) was used fordetection. After 45 minutes incubation at room temperature, the platewas washed with PBS/0.1% Tween20 three times and developed using TMBsubstrate. After 10 minutes incubation, the reaction was stopped by 1%H₂SO₄. The OD450 nm was read by SOFTmax program. The read out wasplotted using GraphPadPrism 3.03 software.

6.13 Construction of IgDART Diabodies

IgDART Diabodies were constructed that contain variable regions capableof binding to CD32 and B-cell receptor complex (“BCRC”). The firstdiabody employed an LGGCGGGS (SEQ ID NO: 267) linker between the VHsequences and the Fc sequences of the molecule. The second diabodyemployed either a LEIK linker having the sequence: LEIK (SEQ ID NO: 268)or a TVSS linker having the sequence TVSS (SEQ ID NO: 269). Thesequences of the chains of these diabodies and encoding polynucleotidesare shown below:

H8B5VL-hBCRCVH M48I, M62K_LGGCG3S_hKappa (SEQ ID NO: 270): DIQMTQSPSSLSASVGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA 50 ASTLDSGVPS RFSGSESGTEFTLTISSLQP EDFATYYCLQ YFSYPLTFGG 100 GTKVEIK GGG SGGGG QVQLV QSGAEVKKPGASVKVSCKAS GYTFTSYWMN 150 WVRQAPGQGL EWIGMIDPSD SETHYNQKFK DRVTMTTDTSTSTAYMELRS 200 LRSDDTAVYY CARAMGYWGQ GTTVTVSS LG GCGGGS RTVA APSVFIFPPS250 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS 300TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 343

The H₈B5VL sequences are fused to the hBCRCVH sequences by the linkerGGGSGGGG (SEQ ID NO: 10) located at position 108-115. The hBCRCVHsequences are fused to the Fc sequences by the linker LGGCGGGS (SEQ IDNO: 267) located at position 229-236 (both shown underlined above). Thepolynucleotide encoding the H₈B5VL-hBCRCVH M48I, M62K_LGGCG3S_hKappasequence is:

(SEQ ID NO: 271): gacatccaga tgacccagtc tccatcctcc ttatctgcct ctgtgggaga50 tagagtcacc atcacttgtc gggcaagtca ggaaattagt ggttacttaa 100 gctggctgcagcagaaacca ggcaaggccc ctagacgcct gatctacgcc 150 gcatccactt tagattctggtgtcccatcc aggttcagtg gcagtgagtc 200 tgggaccgag ttcaccctca ccatcagcagccttcagcct gaagattttg 250 caacctatta ctgtctacaa tattttagtt atccgctcacgttcggaggg 300 gggaccaagg tggaaataaa a ggaggcgga tccggaggcg gaggc caggt350 tcagctggtg cagtctggag ctgaggtgaa gaagcctggc gcctcagtga 400aggtctcctg caaggcttct ggttacacct ttaccagcta ctggatgaac 450 tgggtgcgacaggcccctgg acaagggctt gagtggatcg gaatgattga 500 tccttcagac agtgaaactcactacaatca aaagttcaag gacagagtca 550 ccatgaccac agacacatcc acgagcacagcctacatgga gctgaggagc 600 ctgagatctg acgacacggc cgtgtattac tgtgcgagagctatgggcta 650 ctgggggcaa gggaccacgg tcaccgtctc ctca ctggga ggctgcggcg700 gagggagc cg aactgtggct gcaccatcgg tcttcatctt cccgccatct 750gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa 800 cttctatcccagagaggcca aagtacagtg gaaggtggat aacgccctcc 850 aatcgggtaa ctcccaggagagtgtcacag agcaggacag caaggacagc 900 acctacagcc tcagcagcac cctgacgctgagcaaagcag actacgagaa 1000 acacaaagtc tacgcctgcg aagtcaccca tcagggcctgagctcgcccg 1050 tcacaaagag cttcaacagg ggagagtgtt ag 1082where the sequences encoding the linkers: GGGSGGGG (SEQ ID NO: 10) andLGGCGGGS (SEQ ID NO: 267) are located at position 322-345 and 685-708,respectively (both shown underlined above).

HBCRCVL R45N-h8B5VH_LGGCGGGS-hG1 (SEQ ID NO: 272): DVVMTQSPLS LPVTLGQPASISCKSSQSLL DSDGKTYLNW FQQRPGQSPN 50 RLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP 100 LTFGGGTKLE IK GGGSGGGG EVQLVESGGG LVQPGGSLRLSCAASGFTFS 150 DAWMDWVRQA PGKGLEWVAE IRNKAKNHAT YYAESVIGRF TISRDDAKNS200 LYLQMNSLRA EDTAVYYCGA LGLDYWGQGT LVTVSS LGGC GGGS ASTKGP 250SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV 300 LQSSGLYSLSSVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT 350 HTCPPCPAPE LLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV 400 KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV 450 SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQVSLTCLVKGFY 500 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF550 SCSVMHEALH NHYTQKSLSL SPGK 574The hBCRCVL sequences are fused to the H₈B5VH sequences by the linkerGGGSGGGG (SEQ ID NO: 10) located at position 113-120. The H₈B5VHsequences are fused to the Fc sequences by the linker LGGCGGGS (SEQ IDNO: 267) located at position 237-244 (both shown underlined above). Thepolynucleotide encoding the HBCRCVL R45N-h8B5VH_LGGCGGGS-hG1 sequenceis:

(SEQ ID NO: 273): gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca50 gccggcctcc atctcctgca agtcaagtca gagcctctta gatagtgatg 100 gaaagacatatttgaattgg tttcagcaga ggccaggcca atctccaaac 150 cgcctaattt atctggtgtctaaactggac tctggggtcc cagacagatt 200 cagcggcagt gggtcaggca ctgatttcacactgaaaatc agcagggtgg 250 aggctgagga tgttggggtt tattactgct ggcaaggtacacattttccg 300 ctcacgttcg gcggagggac caagcttgag atcaaa ggag gcggatccgg350 aggcggaggc gaagtgcagc ttgtggagtc tggaggaggc ttggtgcaac 400ctggaggatc cctgagactc tcttgtgccg cctctggatt cacttttagt 450 gacgcctggatggactgggt ccgtcaggcc ccaggcaagg ggcttgagtg 500 ggttgctgaa attagaaacaaagctaaaaa tcatgcaaca tactatgctg 550 agtctgtgat agggaggttc accatctcaagagatgacgc caaaaacagt 600 ctgtacctgc aaatgaacag cttaagagct gaagacactgccgtgtatta 650 ctgtggggct ctgggccttg actactgggg ccaaggcacc ctggtgaccg700 tctccagc ct gggaggctgc ggcggaggga gc gcctccac caagggccca 750tcggtcttcc ccctggcacc ctcctccaag agcacctctg ggggcacagc 800 ggccctgggctgcctggtca aggactactt ccccgaaccg gtgacggtgt 850 cgtggaactc aggcgccctgaccagcggcg tgcacacctt cccggctgtc 900 ctacagtcct caggactcta ctccctcagcagcgtggtga ccgtgccctc 950 cagcagcttg ggcacccaga cctacatctg caacgtgaatcacaagccca 1000 gcaacaccaa ggtggacaag agagttgagc ccaaatcttg tgacaaaact1050 cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt 1100cttcctcttc cccccaaaac ccaaggacac cctcatgatc tcccggaccc 1150 ctgaggtcacatgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1200 aagttcaact ggtacgtggacggcgtggag gtgcataatg ccaagacaaa 1250 gccgcgggag gagcagtaca acagcacgtaccgtgtggtc agcgtcctca 1300 ccgtcctgca ccaggactgg ctgaatggca aggagtacaagtgcaaggtc 1350 tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa1400 agggcagccc cgagaaccac aggtgtacac cctgccccca tcccgggatg 1450agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1500 cccagcgacatcgccgtgga gtgggagagc aatgggcagc cggagaacaa 1550 ctacaagacc acgcctcccgtgctggactc cgacggctcc ttcttcctct 1600 acagcaagct caccgtggac aagagcaggtggcagcaggg gaacgtcttc 1650 tcatgctccg tgatgcatga ggctctgcac aaccactacacgcagaagag 1700 cctctccctg tctccgggta aa 1722where the sequences encoding the linkers: GGGSGGGG (SEQ ID NO: 10) andLGGCGGGS (SEQ ID NO: 267) are located at position 337-360 and 709-732,respectively (both shown underlined above).

H8B5VL-HBCRCVH M48I, M62K_(-4)LEIK_hKappa (SEQ ID NO: 274): DIQMTQSPSSLSASVGDRVT ITCRASQEIS GYLSWLQQKP GKAPRRLIYA 50 ASTLDSGVPS RFSGSESGTEFTLTISSLQP EDFATYYCLQ YFSYPLTFGG 100 GTKVEIK GGG SGGGG QVQLV QSGAEVKKPGASVKVSCKAS GYTFTSYWMN 150 WVRQAPGQGL EWIGMIDPSD SETHYNQKFK DRVTMTTDTSTSTAYMELRS 200 LRSDDTAVYY CARAMGYWGQ GTTV LEIK RT VAAPSVFIFP PSDEQLKSGT250 ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL 300TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 335The H₈B5VL sequences are fused to the HBCRCVH sequences by the linkerGGGSGGGG (SEQ ID NO: 10) located at position 108-115. The HBCRCVHsequences are fused to the Fc sequences by the linker LEIK (SEQ ID NO:268) located at position 225-228 (both shown underlined above). Thepolynucleotide encoding the H₈B5VL-HBCRCVH M48I, M62K_(−4)LEIK_hKappasequence is:

(SEQ ID NO: 275): gacatccaga tgacccagtc tccatcctcc ttatctgcct ctgtgggaga50 tagagtcacc atcacttgtc gggcaagtca ggaaattagt ggttacttaa 100 gctggctgcagcagaaacca ggcaaggccc ctagacgcct gatctacgcc 150 gcatccactt tagattctggtgtcccatcc aggttcagtg gcagtgagtc 200 tgggaccgag ttcaccctca ccatcagcagccttcagcct gaagattttg 250 caacctatta ctgtctacaa tattttagtt atccgctcacgttcggaggg 300 gggaccaagg tggaaataaa a ggaggcgga tccggaggcg gaggc caggt350 tcagctggtg cagtctggag ctgaggtgaa gaagcctggc gcctcagtga 400aggtctcctg caaggcttct ggttacacct ttaccagcta ctggatgaac 450 tgggtgcgacaggcccctgg acaagggctt gagtggatcg gaatgattga 500 tccttcagac agtgaaactcactacaatca aaagttcaag gacagagtca 550 ccatgaccac agacacatcc acgagcacagcctacatgga gctgaggagc 600 ctgagatctg acgacacggc cgtgtattac tgtgcgagagctatgggcta 650 ctgggggcaa gggaccacgg tc ctggagat caag cgaact gtggctgcac700 catcggtctt catcttcccg ccatctgatg agcagttgaa atctggaact 750gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt 800 acagtggaaggtggataacg ccctccaatc gggtaactcc caggagagtg 850 tcacagagca ggacagcaaggacagcacct acagcctcag cagcaccctg 900 acgctgagca aagcagacta cgagaaacacaaagtctacg cctgcgaagt 950 cacccatcag ggcctgagct cgcccgtcac aaagagcttcaacaggggag 1000 agtgt 1005where the sequences encoding the linkers: GGGSGGGG (SEQ ID NO: 10) andLEI K (SEQ ID NO: 268) are located at position 322-345 and 673-684,respectively (both shown underlined above).

HBCRCVL R45N-h8B5VH_(-4)TVSS-hG1 = HBCRCVL R45N- h8B5VH_-hGI (SEQ ID NO:276): DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTYLNW FQQRPGQSPN 50RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP 100 LTFGGGTKLE IKGGGSGGGG  EVQLVESGGG LVQPGGSLRL SCAASGFTFS 150 DAWMDWVRQA PGKGLEWVAEIRNKAKNHAT YYAESVIGRF TISRDDAKNS 200 LYLQMNSLRA EDTAVYYCGA LGLDYWGQGT LVTVSS ASTK GPSVFPLAPS 250 SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFPAVLQSSGLYS 300 LSSVVTVPSS SLGTQTYICN VNHKFSNTKV DKRVEPKSCD KTHTCPPCPA350 PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG 400VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP 450 IEKTISKAKGQPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW 500 ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA 550 LHNHYTQKSL SLSPGK 566The HBCRCVL sequences are fused to the h8B5VH sequences by the linkerGGGSGGGG (SEQ ID NO: 10) located at position 113-120. The h8B5VHsequences are fused to the Fc sequences by the linker TVSS (SEQ ID NO:269) located at position 233-236 (both shown underlined above). Thepolynucleotide encoding the HBCRCVL R45N-h8B5VH_(−4)TVSS-hG1 is:

(SEQ ID NO: 277): gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca50 gccggcctcc atctcctgca agtcaagtca gagcctctta gatagtgatg 100 gaaagacatatttgaattgg tttcagcaga ggccaggcca atctccaaac 150 cgcctaattt atctggtgtctaaactggac tctggggtcc cagacagatt 200 cagcggcagt gggtcaggca ctgatttcacactgaaaatc agcagggtgg 250 aggctgagga tgttggggtt tattactgct ggcaaggtacacattttccg 300 ctcacgttcg gcggagggac caagcttgag atcaaa ggag gcggatccgg350 aggcggaggc  gaagtgcagc ttgtggagtc tggaggaggc ttggtgcaac 400ctggaggatc cctgagactc tcttgtgccg cctctggatt cacttttagt 450 gacgcctggatggactgggt ccgtcaggcc ccaggcaagg ggcttgagtg 500 ggttgctgaa attagaaacaaagctaaaaa tcatgcaaca tactatgctg 550 agtctgtgat agggaggttc accatctcaagagatgacgc caaaaacagt 600 ctgtacctgc aaatgaacag cttaagagct gaagacactgccgtgtatta 650 ctgtggggct ctgggccttg actactgggg ccaaggcacc ctggtg accg700 tctccagc gc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 750tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga 800 ctacttccccgaaccggtga cggtgtcgtg gaactcaggc gccctgacca 850 gcggcgtgca caccttcccggctgtcctac agtcctcagg actctactcc 900 ctcagcagcg tggtgaccgt gccctccagcagcttgggca cccagaccta 950 catctgcaac gtgaatcaca agcccagcaa caccaaggtggacaagagag 1000 ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca1050 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa 1100ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg 1150 acgtgagccacgaagaccct gaggtcaagt tcaactggta cgtggacggc 1200 gtggaggtgc ataatgccaagacaaagccg cgggaggagc agtacaacag 1250 cacgtaccgt gtggtcagcg tcctcaccgtcctgcaccag gactggctga 1300 atggcaagga gtacaagtgc aaggtctcca acaaagccctcccagccccc 1350 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt1400 gtacaccctg cccccatccc gggatgagct gaccaagaac caggtcagcc 1450tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1500 gagagcaatgggcagccgga gaacaactac aagaccacgc ctcccgtgct 1550 ggactccgac ggctccttcttcctctacag caagctcacc gtggacaaga 1600 gcaggtggca gcaggggaac gtcttctcatgctccgtgat gcatgaggct 1650 ctgcacaacc actacacgca gaagagcctc tccctgtctccgggtaaa 1698where the sequences encoding the linkers: GGGSGGGG (SEQ ID NO: 10) andTVSS (SEQ ID NO: 269) are located at position 337-360 and 697-708,respectively (both shown underlined above).

6.14 Optimization of Linkers

As discussed above, the IgDART diabodies of the present inventionpreferably contain linkers between the VH sequences and the Fc sequencesof the molecule. Experiments were conducted to optimize the linkers inorder to maximize yield and activity. The following linkers wereemployed.

SEQ ID NO Linker 278 FNRGECGGGS 279 FNRGECLQVYYRM 280 LEGEEG 281 LEGEEGC282 LEIK 283 LGEEG 284 LGEEGC 285 LGGCGGGS 286 LGKKG 287 LGKKGC 288LKGKKG 289 LKGKKGC 290 LQVYYRM 291 LQVYYRMC 292 TVSS 293 VEPKSCGGGS 294VEPKSCYLYLRARV 295 VQVHYRM 296 VQVHYRMC 297 YLYLRARV 298 YLYLRARVC

The above linkers were introduced into plasmids in order to make a setof IgDART Diabodies having different combinations of linkers:

Chain A Chain B Purified Linker Linker protein Plasmid SEQ SEQ ELISA(After SEC) pMGX ID NO pEE13.4 ID NO pEE6.4 (μg/ml) (1 liter) 900 285 ✓285 ✓ 0.799 0.3 mg 901 283 ✓ 286 ✓ 0.628 0.4 mg 902 284 ✓ 287 ✓ 0.8960.47 mg  903 280 ✓ 288 ✓ 0.557 904 281 ✓ 289 ✓ 0.450 0.4 mg 905 293 ✓278 ✓ 0.360 906 294 ✓ 279 ✓ N/A 907 282 ✓ 292 ✓ N/A 908 297 ✓ 295 ✓0.428 0.2 mg 909 297 ✓ 290 ✓ 0.305 0.3 mg 910 298 ✓ 296 ✓ N/A 911 298 ✓291 ✓ 0.218

The aggregation properties of the produced IgDARTS was determined.

IgDART Total Protein Oligo- Mono- Frag- SB Linkers (mg) mer % mer % ment% Rank 900A/900B 0.51 12 45 43 4 901A/901B 0.72 5 83 12 1 902A/902B 0.7821 48 31 4 903A/903B 0.5 3 84 13 1 904A/904B 0.66 16 65 26 4 905A/905B0.5 20 60 20 3 908A/908B 0.5 13 65 17 2 908A/909B 0.38 22 50 28 3910A/911B 0.2 45 10 45 5

The data unexpectedly showed that constructs having linkers, such asthose employed in 901A/901B; 903A/903B; and 908A/908B gave dramaticallysuperior results (less oligomerization and/or less fragment production)than constructs having linkers, such as 910A/911B.

6.15 E-Coil/K-Coil DARTs

As will be appreciated in view of the foregoing, the individualpolypeptides of a bispecific DART can form two species of homodimers andone species of heterodimer. In one embodiment of the present invention,a charged polypeptide can be added to the C-terminus of one, or morepreferably, both DART polypeptides. By selecting charged polypeptides ofopposite charge for the individual polypeptides of the bispecific DART,the inclusion of such charged polypeptides favors formation ofheterodimers and lessens formation of homodimers. Preferably, apositively charged polypeptide will contain a substantial content ofarginine, glutamine, histidine and/or lysine (or mixtures of such aminoacids) and a negatively charged polypeptide will contain a substantialcontent of aspartate or glutamate (or a mixture of such amino acids).Positively charged polypeptides containing a substantial content oflysine and negatively charged polypeptides containing a substantialcontent of glutamate are particularly preferred. In order to maximizethe electrostatic attraction between such opposingly chargedpolypeptides, it is preferred to employ polypeptides capable ofspontaneously assuming a helical conformation.

Thus, in a preferred embodiment, a positively charged, “E-coil” will beappended to one of the polypeptides being used to form a bispecific DARTand a negatively charged “K-coil” will be appended to the second of theDART's polypeptides (FIG. 37).

particularly preferred E-coil will have the sequence: (EVAALEK)₄:

EVAALEKEVAALEKEVAALEKEVAALEK SEQ ID NO: 299

A particularly preferred K-coil will have the sequence: (KVAALKE)₄:

KVAALKEKVAALKEKVAALKEKVAALKE SEQ ID NO: 300

A preferred DART polypeptide possessing such an E-coil will have thegeneral sequence: [VL Domain]-[GGGSGGGG]-[VH Domain]-[(EVAALEK)₄]-GGGNS,where VL is the DART's variable light Ig domain, GGGSGGGG is SEQ ID NO:10, VH is the DART's variable heavy Ig domain, (EVAALEK)₄ is SEQ ID NO:299, and GGGNS is SEQ ID NO: 301. A preferred DART polypeptidepossessing such a K-coil will have the general sequence: [VLDomain]-[GGGSGGGG]-[VH Domain]-[(KVAALICE)₄]-GGGNS, where VL is theDART's variable light Ig domain, GGGSGGGG is SEQ ID NO: 10, VH is theDART's variable heavy Ig domain, (KVAALKE)₄ is SEQ ID NO: 300, and GGGNSis SEQ ID NO: 301.

6.16 E-Coil/K-Coil Fc-Containing DARTs

In a further embodiment, Fc-regions can be linked to the E and/or Kcoils of E-coil or K-coli DARTs.

Furthering the separation between the Fc regions and the DART VH domainof an Fc-containing DART is desirable in cases in which a less separatedarrangement of such domains results in diminished interaction betweensuch domains and their binding ligands or otherwise interferes with DARTassembly. Although separators of any amino acid sequence may beemployed, it is preferable to employ separators that form an a helixcoils, so as to maximally extend and project the Fc domain away from thevariable domains (FIG. 37). Because the above-described coiledpolypeptides of opposing charge additionally function to promoteheterodimer formation, such molecules are particularly preferredseparators. Such coil-containing Fc-DART molecules provide benefitssimilar to those of Fc-DARTS, including improved serum half-life andeffector function recruitment. The above-described E-coil and K-coilpolypeptides are particularly preferred for this purpose.

Thus, in a preferred embodiment, the E-coil Fc-containing DART will havethe general sequence: [VL Domain]-[GGGSGGGG]-[VHDomain]-[(EVAALEK)₄]-GGG-Fc domain starting with D234 (Kabat numbering),where VL is the DART's variable light Ig domain, GGGSGGGG is SEQ ID NO:10, VH is the DART's variable heavy Ig domain and (EVAALEK)₄ is SEQ IDNO: 299.

Similarly, in a preferred embodiment, the K-coil Fc-containing DART willhave the general sequence: [VL Domain]-[GGGSGGGG]-[VHDomain]-[(KVAALKE)₄]-GGG-Fc domain starting with D234 (Kabat numbering),where VL is the DART's variable light Ig domain, GGGSGGGG is SEQ ID NO:10, VH is the DART's variable heavy Ig domain and (KVAALKE)₄ is SEQ IDNO: 300.

As indicated above, a coil-containing DART molecule or a coil-containingFc-containing DART molecule may contain only a single such coilseparator, or it may contain more than one such separators (e.g., twoseparators, preferably of opposite charge, of which one is linked toeach of the VH domain of the DART's polypeptides). By linking the Fcregion to such separator molecule(s), the ability to make bivalent,tetravalent, etc. versions of the Fc-DART molecules by chain swapping isenhanced (FIG. 39). As shown in FIG. 39, Fc-DART molecules can beproduced that form monomers or dimers depending upon whether the Fcdomain is linked to one or both of the DART VH domains.

6.17 Functional Activity of E-Coil/K-Coil Fc-Containing DARTs

E-coil and/or K-coil Fc-DART species were produced from bi-specific DARTmolecules having: (1) the variable light and heavy regions of the CD79b(BCR complex)-reactive antibody, CB3 and (2) the variable light andheavy regions of a low affinity variant (termed “YA” Variant) of theCD32B-reactive antibody, 2B6. This light chain variable region of thisantibody differs from that of antibody 2B6 in containing mutations: N50Yand V51A. Thus, antibody YA2B6 has a light chain variable regionsequence:

(SEQ ID NO: 302) EIVLTQSPDFQSVTPKEKVTITCRTSQSIGTNIHWYQQKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNTWPFTFGG GTKVEIK.

The sequence of the heavy chain variable region of this antibody is:

(SEQ ID NO: 303) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEW MGVIDPSDTYPNYNKKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARNGDSDYYSGMDYWGQGTTVTVSS;or

The low affinity antibody was selected since it will preferentially bindCD32B in cis on cells expressing CD79b (B cells). As such, theconfiguration will diminish interaction with other CD32B-expressingcells (monocytes, endothelial cells, liver) as well as the undesirabletrans interaction

E-coil and/or K-coil derivatives and E-coil and/or K-coil Fc-containingderivatives of such h2B6YAhCB3 DARTS were made. Size exclusionchromatography was used to analyze the approximate size andheterogeneity of the produced molecules. As shown in FIG. 40, dimerswere formed from E-coil/K-coil DARTS having a single linked Fc regionlinked to an K-coil domain as well as to E-coil/K-coil DARTS having asingle linked Fc region linked to an E-coil domain. Desired monomer, aswell as dimer molecules were recovered from preparations in which Fcregions were linked to both the E and K coils of the same DART molecule,with the monomer being the majority product formed. FIG. 41 shows thepossible structure of the produced dimer molecules.

The size exclusion chromatography fractions were analyzed usingSDS-polyacrylamide gel electrophoresis to further analyze the structuresof the produced molecules (FIG. 42). E-coil/K-coil DART derivatives (noFc region) migrated as two predominant bands each of approximately 28 kD(corresponding to the KFc-containing polypeptide and slightly smallerEFc-containing polypeptide) and a less prominent band at approximately49 kD (corresponding to the E-coil/K-coil DART). The monomer fractionsof the E-coil/K-coil Fc-containing DART derivatives (EFc/K or E/KFc)from the size exclusion chromatography showed only either the larger orsmaller molecular weight band at approximately 28 kD (corresponding towhether the DART was the KFc-containing DART (larger molecular weightband) or the EFc-containing DART (smaller molecular weight band).Material predominantly migrated at approximately 49 kD (corresponding tothe E-coil/K-coil DART). Significant higher molecular weight bands werealso observed.

A bispecific binding ELISA was preformed to characterize the producedmolecules. CD79 was put down on an ELISA plate. DARTs were then bound tothe plate. DART binding was detected using sCD32B-biotin followed byincubation with streptavidin-HRP. As shown in FIG. 43, E-coil/K-coilFc-containing h2B6YAhCB3 DART derivatives (EFc/K or E/KFc) showedsignificant enhancement of binding relative to a h2B6YAhCB3 DART, or toan EFc/KFc h2B6YAhCB3 DART derivative.

The cross-linking of antibodies that have bound to CD79b leads to B cellactivation (Van Kooten, C. et al. (1997) “Cross-Linking Of AntigenReceptor Via Ig-B (B29, CD79b) Can Induce Both Positive And NegativeSignals In CD40-Activated Human B Cells,” Clin. Exp. Immunol.110:509-515). Since the h2B6YAhCB3 DART molecules are capable of bindingto both CD79b and the CD32B inhibitory receptor, they have the abilityto “recruit” CD32B to sites of CD79b binding, and to thereby block Bcell proliferation. To demonstrate this ability, DARTS were incubatedwith B cells that had been exposed to antibodies capable of crosslinkingbound anti-CD79b antibodies. The results of this experiment are shown inFIG. 44. The results show that antibodies directed solely against CD79bor CD32B (Ch2B6N297Q and ChCB3.1N297Q, respectively) failed to inhibit Bcell proliferation. EFc/KFc h2B6YA×hCB3 DART derivatives weresubstantially more effective in inhibiting B cell proliferation, as wash2B6YA×hCB3 DART itself and the h2B6YA×hCB3 VF control. E-coil/K-coilDARTS having only a single linked Fc region (E/KFc h2B6YA×hCB3 DARTderivatives and EFc/K h2B6YA×hCB3 DART derivatives) were found to exertthe greatest inhibition on B cell proliferation.

6.18 DART Modifications for Altering In Vivo Serum Half-Life

As discussed above, small recombinant antibody molecules such asbispecific single-chain molecules (e.g., possessing a molecular mass ofapproximately 55 kDa) are rapidly cleared from circulation. in vivopharmacokinetic studies of DART molecules in mice showed the expectedshort terminal half life of approximately 2 hours.

In some embodiments, such as in the treatment of an acute inflammatorycondition, such short half-life is desired, however, in otherembodiments such as in the treatment of cancer and chronic diseases andconditions, it is preferred for the DART molecules of the presentinvention to exhibit longer half-lives.

In order to improve the in vivo pharmacokinetic properties of DARTmolecules for such uses, DART molecules may be modified to contain apolypeptide portion of a serum-binding protein at one or more of thetermini of the DART molecule. Most preferably, such polypeptide portionof a serum-binding protein will be installed at the C-terminus of theDART molecule. A particularly preferred polypeptide portion of aserum-binding protein for this purpose is the albumin-binding domain(ABD) from streptococcal protein G. The albumin-binding domain 3 (ABD3)of protein G of Streptococcus strain G148 is particularly preferred.

The albumin-binding domain 3 (ABD3) of protein G of Streptococcus strainG148 consists of 46 amino acid residues forming a stable three-helixbundle and has broad albumin binding specificity (Johansson, M. U. etal. (2002) “Structure, Specificity, And Mode Of Interaction ForBacterial Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114-8120).Albumin is the most abundant protein in plasma and has a half-life of 19days in humans. Albumin possesses several small molecule binding sitesthat permit it to non-covalently bind to other proteins and therebyextend their serum half-lives.

To demonstrate the ability of a polypeptide portion of a serum proteinto extend the half-life of a DART, the ABD3 domain of streptococcalprotein G was fused to a recombinant bispecific DART (immunoreactivewith hCD16 and hCD32B antigens) to generate a recombinant antibodymolecule, hCD16-hCD32B ABD-DART (FIG. 45). This ABD-DART showed specificbinding to both antigens as well as with human serum albumin (HSA) andwas able to retarget effector cells in vitro. Compared with the controlDART, this ABD-DART showed strong increase of serum half-life in mice.This approach can be used as a viable route for increasing the half-lifeof potentially important pharmaceuticals like DART to greater than 90minutes, greater than 2 hours, greater than 5 hours, greater than 10hours, greater than 20 hours, and most preferably, greater than 30hours.

Materials and Methods:

Design and Construction of ABD DART: hCD16-hCD32B ABD DART was madeusing as chain1:

-   -   hCD16VL-G3SG4-hCD32BVH-K coil [(KVAALKE)₄]        where CD16VL denotes the 3G8 CD16VL, G3SG4 denotes SEQ ID NO:        10, hCD32BVH denotes the 2B6 CD32BVH, and (KVAALKE)₄ denotes SEQ        ID NO: 300;        and as chain 2:

hCD32BVL-G3SG4 hCD16VH-GGCGGG-E coil [(EVAALEK)₄]-GGGNS-ABD

where CD32BVL denotes CD32BVL, G3SG4 denotes SEQ ID NO: 10, hCD16VHdenotes CD16VH, GGCGGG is residues 2-7 of SEQ ID NO: 267, E coil[(EVAALEK)₄] is SEQ ID NO: 299, GGGNS is SEQ ID NO: 301, and ABD is:

LAEAKVLANR ELDKYGVSDY YKNLINNAKT VEGVKALID EILAALP (SEQ ID NO: 304)

Accordingly, the sequence of chain 1 (h3G8VL1-G3SG4-h2B6VH4-Kcoil-GGGNS) is:

(SEQ ID NO: 305) DIVMTQSPDS LAVSLGERAT INCKASQSVD FDGDSFMNWY QQKPGQPPKLLIYTTSNLES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQSNEDPY TFGQGTKLEIKGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YWIHWVRQAP GQGLEWIGVIDPSDTYPNYN KKFKGRVTMT VVVSTSTAYM ELRSLRSDDT AVYYCARNGD SDYYSGMDYWGQGTTVTVSS GGCGGGKVAA LKEKVAALKE KVAALKEKVA ALKEGGGNS

A preferred polynucleotide encoding chain 1(h3G8VL1-G3SG4-h2B6VH4-Kcoil-GGGNS) is:

(SEQ ID NO 306) gacatcgtga tgacccaatc tccagactct ttggctgtgt ctctaggggagagggccacc atcaactgca aggccagcca aagtgttgat tttgatggtg atagttttatgaactggtac caacagaaac caggacagcc acccaaactc ctcatctata ctacatccaatctagaatct ggggtcccag acaggtttag tggcagtggg tctgggacag acttcaccctcaccatcagc agcctgcagg ctgaggatgt ggcagtttat tactgtcagc aaagtaatgaagatccgtac acgttcggac aggggaccaa gcttgagatc aaaggaggcg gatccggcggcggaggccag gttcagctgg tgcagtctgg agctgaggtg aagaagcctg gggcctcagtgaaggtctcc tgcaaggctt ctggttacac ctttaccaac tactggatac actgggtgcgacaggcccct ggacaagggc ttgagtggat tggagtgatt gatccttctg atacttatccaaattacaat aaaaagttca agggcagagt caccatgacc gtagtcgtat ccacgagcacagcctacatg gagctgagga gcctgagatc tgacgacacg gccgtgtatt actgtgcgagaaacggtgat tccgattatt actctggtat ggactactgg gggcaaggga ccacggtcaccgtctcctcc ggaggatgtg gcggtggaaa agtggccgca ctgaaggaga aagttgctgctttgaaagag aaggtcgccg cacttaagga aaaggtcgca gccctgaaag agggcggcgggaattct

Accordingly, the sequence of chain 2(h2B6VL5-G3SG4-h3G8VH5-Ecoil-GGGNS-ABD) is:

(SEQ ID NO: 307) EIVLTQSPDF QSVTPKEKVT FTCRTSQSIG TNIHWYQQKP DQSPKLLIKEVSESISGVPS RFSGSGSGTD FTLTINSLEA EDAATYYCQQ SNTWPFTFGG GTKVEIKGGGSGGGGQVTLR ESGPALVKPT QTLTLTCTFS GFSLSTSGMG VGWIRQPPGK ALEWLAHIWWDDDKRYNPAL KSRLTISKDT SKNQVVLTMT NMDPVDTATY YCAQINPAWF AYWGQGTLVTVSSGGCGGGE VAALEKEVAA LEKEVAALEK EVAALEKGGG NSLAEAKVLA NRELDKYGVSDYYKNLINNA KTVEGVKALI DEILAALP

A preferred polynucleotide encoding chain 2(h2B6VL5-G3SG4-h3G8VH5-Ecoil-GGGNS-ABD) is:

(SEQ ID NO: 308) gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaaggagaaagtcacc ttcacctgca ggaccagtca gagcattggc acaaacatac actggtaccagcagaaacca gatcagtctc caaagctcct catcaaggag gtttctgagt ctatctctggagtcccatcg aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatagcctggaagct gaagatgctg caacgtatta ctgtcaacaa agtaatacct ggccgttcacgttcggcgga gggaccaagg tggagatcaa aggaggcgga tccggcggcg gaggccaggttaccctgaga gagtctggcc ctgcgctggt gaagcccaca cagaccctca cactgacttgtaccttctct gggttttcac tgagcacttc tggtatgggt gtaggctgga ttcgtcagcctcccgggaag gctctagagt ggctggcaca catttggtgg gatgatgaca agcgctataatccagccctg aagagccgac tgacaatctc caaggatacc tccaaaaacc aggtagtcctcacaatgacc aacatggacc ctgtggatac tgccacatac tactgtgctc aaataaaccccgcctggttt gcttactggg gccaagggac tctggtcact gtgagctccg gaggatgtggcggtggagaa gtggccgcac tggagaaaga ggttgctgct ttggagaagg aggtcgctgcacttgaaaag gaggtcgcag ccctggagaa aggcggcggg aattctctgg ccgaagcaaaagtgctggcc aaccgcgaac tggataaata tggcgtgagc gattattata agaacctgattaacaacgca aagaccgtgg aaggcgtgaa agcactgatt gatgaaattc tggccgccct gcct

Each VL and VH segment was amplified by PCR using hCD16-hCD32B DART astemplate. For chain 2, nucleotide sequences containing E coil and ABDwere formed by primer dimer and then subcloned at the C-terminal end ofVH region of hCD16 using restriction digestion and ligation. Both chainswere cloned into pCIneo vector (Promega, inc.) at NheI-NotII sites. Theindividual plasmids harboring the respective chain1 digested atNgoMIV-NheI and chain2 expression cassettes digested at BstBI-PmeI werethen cloned into a single plasmid for transfection into CHO cells togenerate stable cell lines.

Expression and Purification of Protein: For stable transfection, CHO—Scells were transfected with hCD16-hCD32B EK ABD-DART plasmid DNA. TheABD-DART protein was purified by affinity chromatography using thesoluble version of FcRIIB antigen coupled to CNBr activated Sepharose4B. The concentrated protein was further purified by size exclusionchromatography using Superdex 200HR 10/30.

Binding assay by ELISA: For CD-16 based capture, plates were coated withFcRIIB antigen at a concentration of 2 ug/mL at 4° C. for overnight.Plates were then blocked with 0.5% Peptone in PBS-T. Purified proteinsdiluted in a serial dilution of two fold were bound on plate for 1 h atroom temperature. Finally, detection was performed using biotinylatedCD32B (50 ng/mL) followed by HRP conjugated Streptavidin ( 1/1000,BD-Pharm). HRP activity was measured by addition of TMB and plate wasread in a plate reader at OD 450 nm.

For human serum albumin (HSA) capture, plates were coated with HSA at aconcentration of 2 ug/mL at 4° C. for overnight. After that the sameprocedures were followed to perform the dual affinity ELISA.

Peripheral-blood mononuclear cell-mediated ADCC assay: Cytotoxicity wasmeasured by LDH release assay. Peripheral blood mononuclear cells (PBMC)were purified from whole human blood (Lonza Walkersville, Inc,Gaithersburg, Md.) by Ficoll-Hypaque (Amersham Biosciences, Piscataway,N.J.) density gradient centrifugation following manufacturer'sinstruction. 2×10⁴ target cells are plated into each well of around-bottom 96-well tissue culture plate. A one to four serial dilutionof different DART or antibody molecules are added to the cells in theplate. After that, 6×10⁵ PBMCs are added to the same wells. Plate isthen incubated for overnight at 37° C. and 5% CO₂ incubator. The plateis then spun at 1200 rpm for 5 minutes, 50 μl of supernatant istransferred to a flat bottom ELISA plate. 50 μl of LDH substratesolution (Promega) is added to each well, and the plate is incubated for30 min in dark at room temperature. Then 50 μl of stop solution is addedto each well, and the plate is read at 490 nm within one hour. Thepercent cytotoxicity of each well is calculated with raw O.D. reading as

(Sample−AICC)/(Target Max−Target Spontaneous)×100

where AICC is the antibody-independent cellular cytotoxicity. The doseresponse curve is generated using Prism software.

Pharmacokinetic Study: C57BI/6 mice were injected with a singleintravenous injection of hCD16-hCD32B DART at 5 mg/kg. Mouse serum wascollected at Pre-dose, 2, 30 min; 1, 3, 6, 24 and 72 h. hCD16-hCD32BDART concentration in serum were quantified. Pharmacokineticcalculations of hCD16-hCD32B DART were performed by means of thepharmacokinetic software package WinNonlin Professional 5.1 (PharsightCorporation, USA). Parameters were determined by non-compartmentalanalysis (NCA). The non-compartmental analysis was based on a model(Model 201) requiring an intravenous injection of the drug. The lineartrapezoidal method was used for parameter calculation.

Results

Expression and binding study by ELISA: The hCD16-hCD32B ABD-DART wasexpressed efficiently at a concentration of 6.5 mg per liter inmammalian CHO—S cells. Binding activity of the purified ABD-DART proteinto the respective antigens was assessed by ELISA. Results showed thathCD16-hCD32B ABD-DART binds simultaneously with both of the antigens,CD16 as well as with CD32B (FIG. 46A). The binding profile coincideswith control hCD16-hCD32B DART protein binding. Affinity of the purifiedhCD16-hCD32B ABD-DART to human serum albumin (HSA) was also demonstratedby ELISA (FIG. 46B). The result showed strong binding of ABD fusion DARTto HSA, whereas no binding was observed with control hCD16-hCD32B DART.

In vitro cytotoxicity of ABD-DART: In order to demonstrate thesimultaneous binding of this bispecific ABD-DART to two antigens, one onthe effector cell and one on a target cell, the redirected cell killingassay was performed. Using human PBMC as effector cells, hCD16-hCD32BABD-DART induced potent, dose-dependent, cytotoxicity against CD32Bpositive B cell lines, Daudi (FIG. 47). The result showed that thepotency of ABD-DART was equivalent to that of parental DART.

Pharmacokinetic Properties of ABD-DART: The pharmacokinetic propertiesof hCD16-hCD32B ABD-DART were analyzed by ELISA of serum samples after asingle dose i.v. injection into C57BI/6 mice (FIG. 48). Both of theproteins, DART and ABD-DART showed biphasic elimination fromcirculation. The PK study of ABD-DART showed a prolonged circulationtime, with an increased terminal half-life of 35.1 h compared to 1.2 hfor regular DART (FIG. 48, Table 17). The improvement of pharmacokineticproperties was also demonstrated by comparison of the area under thecurve (AUC). For the construct ABD-DART the AUC increased by a factor ofalmost 30 after fusion to ABD (Table 17).

TABLE 17 ABD-DART DART T½ (hr) 35.1 1.2 Cmax (μg/mL) 156.3 103.7 Tmax(hr) 0.5 0.033 AUC 4408.2 138.3

In sum, an albumin binding domain fused DART protein (referred to asABD-DART) was successfully designed and produced. The hCD16-hCD32BABD-DART was found to retain the specificities to its two recognizedantigenic determinants: CD16 and CD32B. ABD-DART was found to show highaffinity with human serum albumin. The fusion of ABD did not reduce thebiological activity (i.e., the potency of the DART for redirected tumorcell killing). The fusion of DART molecule to ABD led to a substantialimprovement (increase) in its in vivo half-life, and accomplished thisgoal without a dramatic increase in size. The ability to retain a smallsize is significant and advantageous since it facilitates the ability ofthe DART to diffuse into tumor tissues.

6.19 Her2/B Cell Receptor DARTs

An IgDART Diabody was constructed that contained variable regionscapable of binding to Her2/neu and to the T-cell receptor (“TCR”).

As discussed above, the TCR is natively expressed by CD4+ or CD8+T-cells, and permits such cells to recognize antigenic peptides that arebound and presented by class I or class II MHC proteins ofantigen-presenting cells. Recognition of a pMHC (peptide-MHC) complex bya TCR initiates the propagation of a cellular immune response that leadsto the production of cytokines and the lysis of the antigen-presentingcell. HER2/neu, an important member of the ErbB family, has beenextensively investigated because of its role in several human carcinomasand in mammalian development. (Hynes and Stern (1994) Biochim. etBiophys. Acta 1198:165-184; and Dougall et al. (1994) Oncogene9:2109-2123; Lee et al. (1995) Nature 378:394-398). The human HER2/neugene and HER2/neu protein are described in Semba et al. (1985) Proc.Natl. Acad. Sci. (U.S.A.) 82:6497-6501 and Yamamoto et al. (1986) Nature319:230-234, and the sequence is available in GenBank as accessionnumber X03363. HER2/neu comprises four domains: an extracellular domainto which ligand binds; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues that can be phosphorylated.(Plowman et al. (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90:1746-1750).The sequence of the HER2/neu extracellular (ECD) domain was described byFranklin et al. (2004) Cancer Cell. 5(4):317-328, and is available inProtein DataBank Record 1 S78 (2004).

HER2/neu functions as a growth factor receptor and is often expressed bytumors such as breast cancer, colon cancer, bladder cell cancer, ovariancancer and lung cancer. HER2/neu is overexpressed in 25-30% of humanbreast and ovarian cancers, and is associated with aggressive clinicalprogression and poor prognosis in these patients. (Slamon et al. (1987)Science 235:177-182; Slamon et al. (1989) Science 244:707-712).Overexpression of HER2/neu has also been observed in other carcinomasincluding carcinomas of the stomach, endometrium, salivary gland, lung,kidney, colon, thyroid, pancreas and bladder. (See, e.g., King et al.(1985) Science 229:974; McCann et al. (1990) Cancer 65:88-92; Yonemuraet al. (1991) Cancer Research 51:1034).

A number of monoclonal antibodies and small molecule tyrosine kinaseinhibitors targeting HER-1 or HER2/neu have been developed, including,in particular, a humanized variant of a murine monoclonal antibody knownas 4D5 (HERCEPTIN®, Genentech, Inc.) that recognizes an extracellularepitope (amino acids 529 to 627) in the cysteine-rich II domain ofHER2/neu, which resides very close to the protein's transmembraneregion. Studies have shown that in HER2/neu overexpressing breast cancercells, treatment with antibodies specific to HER2/neu in combinationwith chemotherapeutic agents (e.g., cisplatin, doxoubicin, taxol)elicits a higher cytotoxic response than treatment with chemotherapyalone. (Hancock et al. (1991) Cancer Res. 51:4575-4580; Arteaga et al.(1994) Cancer 54:3758-3765; Pietras et al. (1994) Oncogene 9:1829-1838).One possible mechanism by which HER2/neu antibodies might enhanceresponse to chemotherapeutic agents is through the modulation ofHER2/neu protein expression or by interfering with DNA repair.(Stancovski et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:8691-8695;Bacus et al. (1992) Cell Growth & Diff. 3:401-411; Bacus et al. (1993)Cancer Res. 53:5251-5261; Klapper et al. (1997) Oncogene 14:2099-2109;Klapper et al. (2000) Cancer Res. 60:3384-3388; Arteaga et al. (2001) JClinical Oncology 19(18s):32s-40s. Although in certain cases,anti-HER2/neu antibodies such as HERCEPTIN® provide therapeutic benefitto patients, the majority of breast cancer and other patients exhibitrefractory responses to such antibodies. These responses reflect, inpart, differences in the extent of the overexpression of HER2/neu by thepatient's cancer cells.

As a consequence of containing variable regions capable of binding toHer2/neu and to the T-cell receptor (“TCR”), the DART has the ability tobind to HER2-expressing cells and to thereby attach to such cells adomain capable of binding to the T-cell receptor. When such T cells bindto this domain, they activate to initiate an immune response that leadsto the killing of the HER2-expressing cells.

The amino acid and nucleic acid sequences for such DART are providedbelow, with VL and VH sequences shown in plain text, the VL-VH linkershown in underlined text, and the sequence encoding the C-terminalheterodimerization motif (SEQ ID NO: 313: GFNRGEC or SEQ ID NO: 314:GVEPKSC) shown in bold and italics.

TCRVL-HER2VH amino acid sequence (SEQ ID NO: 315) EIVLTQSPAT LSLSPGERATLSCSATSSVS YMHWYQQKPG KAPKRWIYDT SKLASGVPSR FSGSGSGTEF TLTISSLQPEDFATYYCQQW SSNPLTFGQG TKLEIKGGGS GGGGQVQLQQ SGPELVKPGA SLKLSCTASGFNIKDTYIHW VKQRPEQGLE WIGRIYPTNG YTRYDPKFQD KATITADTSS NTAYLQVSRLTSEDTAVYYC SRWGGDGFYA MDYWGQGASV

TCRVL-HER2VH-encoding nucleic acid sequence (SEQ ID NO: 316) gaaattgtgttgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc ctctcctgcagtgccacctc aagtgtaagt tacatgcact ggtatcagca gaaaccaggg aaagcccctaagcgctggat ctatgacaca tccaaactgg cttctggggt cccatcaagg ttcagcggcagtggatctgg gacagaattt actctcacaa tcagcagcct gcagcctgaa gattttgcaacttattactg tcagcagtgg agtagtaacc cgctcacgtt tggccagggg accaagcttgagatcaaagg aggcggatcc ggcggcggag gccaggttca gctgcagcag tctgggccagagcttgtgaa gccaggggcc tcactcaagt tgtcctgtac agcttctggc ttcaacattaaagacaccta tatacactgg gtgaaacaga ggcctgaaca gggcctggaa tggattggaaggatttatcc tacgaatggt tatactagat atgacccgaa gttccaggac aaggccactataacagcaga cacatcctcc aacacagcct acctgcaggt cagccgcctg acatctgaggacactgccgt ctattattgt tctagatggg gaggggacgg cttctatgct atggactactggggtcaagg agcctcggtc accgtgagct

 

HER2VL-TCRVH amino acid sequence (SEQ ID NO: 317) DIVMTQSHKF MSTSVGDRVSITCKASQDVN TAVAWYQQKP GHSPKLLIYS ASFRYTGVPD RFTGSRSGTD FTFTISSVQAEDLAVYYCQQ HYTTPPTFGG GTKVEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKASGYKFTSYVMH WVRQAPGQGL EWIGYINPYN DVTKYNEKFK GRVTITADKS TSTAYMELSSLRSEDTAVHY CARGSYYDYD GFVYWGQGTL

HER2VL-TCRVH-encoding nucleic acid sequence (SEQ ID NO: 318) gacatcgtgatgacccagtc ccacaagttc atgtccacct ctgtgggcga tagggtcagc atcacctgcaaggccagcca ggatgtgaat actgctgtag cctggtatca gcagaaacca ggacattctcccaaactgct gatttactcc gcatccttcc ggtacactgg agtccctgat cgcttcactggcagcagatc tgggacagat ttcactttca ccatcagcag tgtgcaggct gaagacctggcagtttatta ctgtcagcaa cattatacta cacctcccac cttcggaggg ggtaccaaggtggagatcaa aggaggcgga tccggcggcg gaggccaggt tcagctggtg cagtctggagctgaggtgaa gaagcctggg gcctcagtga aggtctcctg caaggccagc ggttacaagtttaccagcta cgtgatgcac tgggtgcgac aggcccctgg acaagggctt gagtggatcggatatattaa tccttacaat gatgttacta agtacaatga gaagttcaaa ggcagagtcacgattaccgc ggacaaatcc acgagcacag cctacatgga gctgagcagc ctgagatccgaggacacggc cgtgcactac tgtgcgagag ggagctacta tgattacgac gggtttgtttactggggcca agggactctg gtcactgtga

 

In a preferred embodiment, such constructs are modified to contain an Ecoil or K coil domain that facilitates the formation of heterodimers(i.e., TCRVL-HER2VH×HER2VL-TCRVH dimers). The amino acid and nucleicacid sequences for such DART are provided below, with VL and VHsequences shown in plain text, the VL-VH linker shown in underlinedtext, and the sequence encoding Cys-containing linker for dimerization(GGCGGG; residues 2-7 of SEQ ID NO: 267) shown in italics. The E coil ofK coil heterodimerization domain is double-underlined (the preferred“E-coil” sequence is 4 heptameric repeats of EVAALEK; SEQ ID NO: 299;the preferred “K-coil” sequence is 4 heptameric repeats of r KVAALKE(SEQ ID NO: 300). The sequence following the E coil or the K coil has noascribed function.

TCRVL-HER2VH-E coil amino acid sequence (SEQ ID NO: 319) EIVLTQSPATLSLSPGERAT LSCSATSSVS YMHWYQQKPG KAPKRWIYDT SKLASGVPSR FSGSGSGTEFTLTISSLQPE DFATYYCQQW SSNPLTFGQG TKLEIKGGGS GGGGQVQLQQ SGPELVKPGASLKLSCTASG FNIKDTYIHW VKQRPEQGLE WIGRIYPTNG YTRYDPKFQD KATITADTSSNTAYLQVSRL TSEDTAVYYC SRWGGDGFYA MDYWGQGASV

EVAALEKEVA ALEKEVAALE KEVAALEKGG GNS TCRVL-HER2VH-E coil-encodingnucleic acid sequence (SEQ ID NO: 320) gaaattgtgt tgacacagtc tccagccaccctgtctttgt ctccagggga aagagccacc ctctcctgca gtgccacctc aagtgtaagttacatgcact ggtatcagca gaaaccaggg aaagccccta agcgctggat ctatgacacatccaaactgg cttctggggt cccatcaagg ttcagcggca gtggatctgg gacagaatttactctcacaa tcagcagcct gcagcctgaa gattttgcaa cttattactg tcagcagtggagtagtaacc cgctcacgtt tggccagggg accaagcttgagatcaaagg aggcggatcc ggcggcggag gccaggttca gctgcagcag tctgggccagagcttgtgaa gccaggggcc tcactcaagt tgtcctgtac agcttctggc ttcaacattaaagacaccta tatacactgg gtgaaacaga ggcctgaaca gggcctggaa tggattggaaggatttatcc tacgaatggt tatactagat atgacccgaa gttccaggac aaggccactataacagcaga cacatcctcc aacacagcct acctgcaggt cagccgcctg acatctgaggacactgccgt ctattattgt tctagatggg gaggggacgg cttctatgct atggactactgqggtcaagg agcctcggtc accgtgagct ccggaggatg tggcggtggagaagtggccg cactggagaa agaggttgct gctttggaga aggaggtcgc tgcacttgaaaaggaggtcg cagccctgga gaaaggcggc gggaattct HER2VL-TCRVH-K coil aminoacid sequence (SEQ ID NO: 321) DIVMTQSHKF MSTSVGDRVS ITCKASQDVNTAVAWYQQKP GHSPKLLIYS ASFRYTGVPD RFTGSRSGTD FTFTISSVQA EDLAVYYCQQHYTTPPTFGG GTKVEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYKFTSYVMHWVRQAPGQGL EWIGYINPYN DVTKYNEKFK GRVTITADKS TSTAYMELSS LRSEDTAVHYCARGSYYDYD GFVYWGQGTL VTVSSGGCGG GKVAALKEKV AALKEKVAAL KEKVAALKEG GGNSHER2VL-TCRVH-K coil-encoding nucleic acid sequence (SEQ ID NO: 322)gacatcgtga tgacccagtc ccacaagttc atgtccacct ctgtgggcga tagggtcagcatcacctgca aggccagcca ggatgtgaat actgctgtag cctggtatca gcagaaaccaggacattctc ccaaactgct gatttactcc gcatccttcc ggtacactgg agtccctgatcgcttcactg gcagcagatc tgggacagat ttcactttca ccatcagcag tgtgcaggctgaagacctgg cagtttatta ctgtcagcaa cattatacta cacctcccac cttcggagggggtaccaagg tggagatcaa aggaggcgga tccggcggcg gaggccaggt tcagctggtgcagtctggag ctgaggtgaa gaagcctggg gcctcagtga aggtctcctg caaggccagcggttacaagt ttaccagcta cgtgatgcac tgggtgcgac aggcccctgg acaagggcttgagtggatcg gatatattaa tccttacaat gatgttacta agtacaatga gaagttcaaaggcagagtca cgattaccgc ggacaaatcc acgagcacag cctacatgga gctgagcagcctgagatccg aggacacggc cgtgcactac tgtgcgagag ggagctacta tgattacgacgggtttgttt actggggcca agggactctg gtcactgtga gctccggagg atgtggcggt ggaaaagtgg ccgcactgaa ggagaaagtt gctgctttga aagagaaggt cgccgcacttaaggaaaagg tcgcagccct gaaagagggc ggcgggaatt ct

DART molecules having Her2 and T-cell receptor (TCR) binding domainswere tested for their ability to mediate cytotoxicity in multiple breastcancer, colon cancer and bladder cancer cell lines that had beenpreviously characterized as exhibiting low levels of HER2 expression(and thus being refractory to treatment with the anti-Her2/neu antibody,Herceptin®. The tested breast cancer cell lines are ZR75-1 (HER2 2+)(FIG. 49A), MCF-7 (HER2 1+) (FIG. 49B) and MDA-MB468 (HER2-ve) (FIG.49C). The non-breast cancer cell lines tested are HT-29 (colon cancercell line) (FIG. 49D) and SW780 (bladder cancer cell line) (FIG. 49E).As shown in FIG. 49A-E, such DART molecules were substantially moreeffective than HERCEPTIN® in mediating cytotoxicity of tumor-derivedcell lines, both in terms of the concentrations required to achieveequivalent cytotoxicity, and in terms of the maximum levels ofcytotoxicity observed.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. Such modifications areintended to fall within the scope of the appended claims. Allreferences, patent and non-patent, cited herein are incorporated hereinby reference in their entireties and for all purposes to the same extentas if each individual publication or patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

1. A diabody molecule comprising a first polypeptide chain and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, and (iii) a third domain comprising an Fc domain or portion thereof, which first domain and second domain are covalently linked such that the first domain and second domain do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising an Fc domain, which fourth domain and fifth domain are covalently linked such that the fourth domain and fifth domain do not associate to form an epitope binding site; wherein the first domain and the fifth domain associate to form a first binding site (VL1)(VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2)(VH2) that binds the second epitope; and wherein: (1) said second and said third domains; or (2) said fifth and said sixth domains; are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 2. The diabody molecule of claim 1, wherein the third domain further comprises a hinge domain.
 3. A diabody molecule comprising a first polypeptide chain and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, and (iii) a third domain comprising a hinge domain and an Fc domain or portion thereof, which first domain and second domain are covalently linked such that the first domain and second domain do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising the amino acid sequence of at least the C-terminal 2 to 8 amino acid residues of a human light chain constant domain, which fourth domain and fifth domain are covalently linked such that the fourth domain and fifth domain do not associate to form an epitope binding site; wherein the first domain and the fifth domain associate to form a first binding site (VL1)(VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2)(VH2) that binds the second epitope; and wherein: (1) said second and said third domains; or (2) said fifth and said sixth domains; are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 4. A diabody molecule comprising a first polypeptide chain and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, and (iii) a third domain comprising a hinge domain, which first domain and second domain are covalently linked such that the first domain and second domain do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising the amino acid sequence of at least the C-terminal 2 to 8 amino acid residues of a human light chain constant domain, which fourth domain and fifth domain are covalently linked such that the fourth domain and fifth domain do not associate to form an epitope binding site; wherein the first domain and the fifth domain associate to form a first binding site (VL1)(VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2)(VH2) that binds the second epitope; and wherein: (1) said second and said third domains; or (2) said fifth and said sixth domains; are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 5. A diabody molecule comprising a first polypeptide chain and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, and (iii) a third domain comprising an Fc domain or portion thereof, which first domain and second domain are covalently linked such that the first domain and second domain do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), and (ii) a fifth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), which fourth domain and fifth domain are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; wherein the first domain and the fifth domain associate to form a first binding site (VL1)(VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2)(VH2) that binds the second epitope; wherein the third domain is N-terminal to both the first domain and the second domain; and wherein: (1) said second and said third domains; or (2) said fifth and said sixth domains; are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 6. The diabody molecule of claim 5, wherein the third domain further comprises a hinge region.
 7. A diabody molecule comprising a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, and (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, which first domain and second domain are covalently linked such that the first domain and second domain do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a third domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), and (ii) a fourth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), which third domain and fourth domain are covalently linked such that the third domain and fourth domain do not associate to form an epitope binding site; wherein the first domain and the third domain associate to form a first binding site (VL1)(VH1) that binds the first epitope, which epitope binding site is specific for CD32B; wherein the second domain and the fourth domain associate to form a second binding site (VL2)(VH2) that binds the second epitope, which epitope binding site is specific for CD16; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked via a disulfide bond between at least one cysteine residue outside of the first domain and the second domain on the first polypeptide chain and at least one cysteine residue outside of the third domain and the fourth domain on the second polypeptide chain, which cysteine residue on the first polypeptide chain is not at the C-terminus of the first polypeptide chain and cysteine residue on the second polypeptide chain is not at the C-terminus of the second polypeptide chain; and wherein: (1) said second and said third domains; or (2) said fifth and said sixth domains; are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 8. A diabody molecule comprising a first polypeptide chain and a second polypeptide chain, wherein: (A) said first polypeptide chain comprises: (i) a domain (A) comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for an epitope (1); (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2); and (iii) a domain (C) comprising a light chain constant region (CL) domain or portion thereof; (B) said second polypeptide chain comprises: (i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for said epitope (2); (ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) specific for said epitope (1); (iii) a domain (F) comprising a heavy chain constant region 1 (CH1) domain or portion thereof, and (iv) a hinge region a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3); wherein: said domains (A) and (B) do not associate with one another to form an epitope binding site; said domains (D) and (E) do not associate with one another to form an epitope binding site; said domains (A) and (E) associate to form a binding site that binds said epitope (1); said domains (B) and (D) associate to form a binding site that binds said epitope (2); said domains (C) and (F) are associated together via a disulfide bond to form a CH1 domain or a portion thereof; wherein: (1) said domains (B) and (C); or (2) said domains (E) and (F); are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 9. The diabody molecule of claim 8, wherein said molecule additionally comprises a third polypeptide chain and a fourth polypeptide chain, wherein: (A) said third polypeptide chain comprises: (i) a domain (G) comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for an epitope (3); (ii) a domain (H) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (4); and (iii) a domain (I) comprising a light chain constant region (CL) domain or portion thereof; (B) said fourth polypeptide chain comprises: (i) a domain (J) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for said epitope (4); (ii) a domain (K) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) specific for said epitope (3); (iii) a domain (L) comprising a heavy chain constant region 1 (CH1) domain or portion thereof; wherein: said domains (G) and (H) do not associate with one another to form an epitope binding site; said domains (J) and (K) do not associate with one another to form an epitope binding site; said domains (G) and (K) associate to form a binding site that binds said epitope (3); said domains (H) and (J) associate to form a binding site that binds said epitope (4); said domains (I) and (L) are associated together via a disulfide bond to form a CH1 domain or a portion thereof; and said hinge region, heavy chain constant region 2 (CH2) and said heavy chain constant region 3 (CH3) of said second and fourth polypeptide chains associate to form an Fc region; and wherein: (1) said domains (B) and (C); (2) said domains (E) and (F); (3) said domains (H) and (I); or (4) said domains (K) and (L); are linked to one another by an intervening polypeptide linker having a sequence selected from the group consisting of SEQ ID NOs: 278-298.
 10. The diabody of claim 1, 2, 3, 4, 5, 6 or 7 wherein said first or said second polypeptide chains of said diabody additionally comprise an E-coil or a K-coil separator.
 11. The diabody of claim 10 wherein both said first and second polypeptide chains of said diabody comprise an E-coil or a K-coil separator.
 12. The diabody of claim 10 wherein said E-coil or a K-coil separator is linked to an Fc fragment.
 13. The diabody of claim 11 wherein said E-coil or a K-coil separator is linked to an Fc fragment.
 14. The diabody of claim 10, wherein said E-coil or a K-coil separator is linked to a polypeptide portion of a serum-binding protein, said polypeptide portion being capable of binding to said serum protein.
 15. The diabody of claim 14, wherein said serum-binding protein is an albumin binding protein.
 16. The diabody of claim 15, wherein said albumin binding protein is streptococcal protein G and said polypeptide is an albumin-binding domain (ABD) of said streptococcal protein G.
 17. The diabody of claim 16, wherein albumin-binding domain (ABD) of said streptococcal protein G is albumin-binding domain 3 (ABD3) of protein G of Streptococcus strain G148.
 18. A diabody exhibiting an in vivo serum half-life greater than 2 hours.
 19. The diabody of claim 18, wherein said diabody exhibits an in vivo serum half-life greater than 10 hours.
 20. The diabody of claim 18, wherein said diabody exhibits an in vivo serum half-life greater than 20 hours.
 21. A DART molecule having a domain that is a binding ligand for the Natural Killer Group 2D (NKG2D) receptor.
 22. The DART molecule of claim 21, wherein said molecule additionally binds to a tumor-associated antigen.
 23. The DART molecule of claim 22, wherein said tumor-associated antigen is a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B cell malignancy, an antigen associated with multiple myeloma, an antigen associated with non-Hodgkins lymphoma, or an antigen associated with chronic lymphocytic leukemia.
 24. The DART molecule of claim 22, wherein said tumor-associated antigen is A33; ADAM-9; ALCAM; B1; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD5; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD32B; CD36; CD40; CD45; CD46; CD56; CD79a; CD79b; CD103; CD154; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; an Ephrin receptor; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2; GD3; GM2; gp100; HER-2/neu; human papillomavirus-E6; human papillomavirus-E7; Integrin Alpha-V-Beta-6; JAM-3; KID3; KID31; KSA (17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M (Oncostatin Receptor Beta); p15; PIPA; PSA; PSMA; RAAG10; ROR1; SART; sTn; TEST; the TNF-α receptor; the TNF-β receptor; the TNF-γ receptor; the Transferrin Receptor or the VEGF receptor.
 25. The DART molecule of claim 24, wherein said tumor-associated antigen is HER-2/neu.
 26. A DART molecule having T-cell receptor (TCR) binding domain.
 27. The DART molecule of claim 26, wherein said molecule additionally binds to a tumor-associated antigen.
 28. The DART molecule of claim 27, wherein said tumor-associated antigen is a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B cell malignancy, an antigen associated with multiple myeloma, an antigen associated with non-Hodgkins lymphoma, or an antigen associated with chronic lymphocytic leukemia.
 29. The DART molecule of claim 27, wherein said tumor-associated antigen is A33; ADAM-9; ALCAM; B1; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD5; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD32B; CD36; CD40; CD45; CD46; CD56; CD79a; CD79b; CD103; CD154; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; an Ephrin receptor; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2; GD3; GM2; gp100; HER-2/neu; human papillomavirus-E6; human papillomavirus-E7; Integrin Alpha-V-Beta-6; JAM-3; KID3; KID31; KSA (17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyl-transferase; Oncostatin M (Oncostatin Receptor Beta); p15; PIPA; PSA; PSMA; RAAG10; ROR1; SART; sTn; TEST; the TNF-α receptor; the TNF-β receptor; the TNF-γ receptor; the Transferrin Receptor or the VEGF receptor.
 30. The DART molecule of claim 29, wherein said tumor-associated antigen is HER-2/neu. 